Method for typing of HLA alleles

ABSTRACT

The present invention relates to the typing of HLA alleles. The sequence of exon 2 and exon 3 of the alleles HLA-B*3913, HLA-B*1406, and HLA-B*51new and of exon 2 of the alleles HLA-DRB1*0820, HLA-DRB1*04new and HLA-DRB4*01new are disclosed. The present invention relates to methods for typing of said alleles. According to a preferred embodiment, said typing comprises the following steps: i) amplifying a relevant fragment of said alleles using at least one suitable pair of primers; ii) hybridizing the amplification product of step i) to at least one probe that specifically hybridizes to a target region comprising one or more polymorphic nucleotides in said relevant fragment; iii) determining from the result of step ii) the absence or presence of said alleles in the sample. The present invention further provides primers and probes to be used in said methods for typing. A diagnostic kit comprising said primers and probes is also part of the present invention.

FIELD OF THE INVENTION

[0001] This invention relates to the typing of human leukocyte antigen(HLA) alleles. More particularly, the present invention relates to thetyping of new HLA alleles.

BACKGROUND OF THE INVENTION

[0002] The human major histocompatibility complex (MHC) is containedwithin about 4 Mbp of DNA on the short arm of chromosome 6 at 6p21.3(Campbell and Trowsdale, 1993). The human MHC is divided into class I,class II and class III regions. The genes of class I and class II encodehighly polymorphic cell-surface molecules that bind and presentprocessed antigens in the form of peptides to T-lymphocytes, initiatingboth cellular and humoral immune responses. The class I molecules,HLA-A, -B, and -C, are found on most nucleated cells. They arecell-surface glycoproteins that bind and present processed peptidesderived from endogenously synthesized proteins to CD8+ T-cells. Theseheterodimers consist of an HLA-encoded α-chain associated with a non-MHCencoded monomorphic polypeptide, β₂-microglobulin (Townsend and Bodmer,1989, Spencer and Parham, 1996). The class II molecules are encoded inthe HLA-D region. These cell-surface glycoproteins consist ofHLA-encoded α-, and β-chains, associated as heterodimers on the cellsurface of antigen-presenting cells such as B-cells and macrophages.Class II molecules serve as receptors for processed peptides. However,these peptides are derived predominantly from membrane and extracellularproteins and are presented to CD4+ T-cells. The HLA-D region containsseveral class II genes and has three main subregions: HLA-DR, -DQ, and-DP. Both the HLA-DQ and -DP regions contain one functional gene foreach of their α- and β-chains. The HLA-DR subregion contains onefunctional gene for the α-chain; the number of functional genes for theβ-chain varies from one to two according to the haplotype (Andersson etal., 1987; Apple and Erlich, 1996).

[0003] A variety of techniques are currently used to detect HLApolymorphism, including serological, biochemical, T-cell recognitionand, most recently, molecular biological methods. Serology remains themainstay method for HLA typing—especially for class I—for many routinehistocompatibility laboratories. The micro-lymphocytotoxicity assay(Kissmeyer et al., 1969; Terasaki and McClelland, 1964) is the standardapproach: viable peripheral blood mononuclear cells (class I) orseparate B-cells (class II) are mixed with antisera (polyclonal ormonoclonal) of known HLA specificity.

[0004] Detection of polymorphism can be achieved by looking at thedifferent amino acid composition of HLA molecules through biochemicaltechniques such as one-dimensional isoelectric focusing (IEF; Yang,1987). This method relies on amino acid substitutions contributing tochanges in charge of the HLA molecule.

[0005] Another HLA typing method is the mixed lymphocyte reaction (MLR).Concurrent to observations being made using HLA-specific antisera, itwas noted that lymphocytes from two unrelated sources, when mixed inculture, would proliferate (Hirschorn et al., 1963).

[0006] Analysis of HLA specificities from DNA provided a new approach todefining their polymorphic differences. Rather than looking atdifferences in the expressed molecule, polymorphism is characterized atthe nucleotide level.

[0007] An important and powerful development in the field of molecularbiology has been the polymerase chain reaction (PCR, Mullis et al.,1986; Mullis and Faloona, 1987). In tissue typing, PCR is used toamplify the polymorphic regions of HLA genes. This HLA PCR product canthen be analysed for its polymorphic differences, to establish thetissue type. A number of such approaches have been developed, includinghetero duplex analysis of PCR products (Clay et al., 1994),single-stranded conformational polymorphism analysis of the PCR product(PCR-SSCP; Yoshida et al., 1992), sequence-based typing (SBT; Santamariaet al., 1992 and 1993), the use of sequence specific primers in PCRreaction (PCR-SSP; Olerup and Zetterquist, 1991), the use of PCR incombination with sequence-specific oligonucleotide probing (PCR-SSOP;Saiki et al., 1986) or probing by reverse dot-blot (Saiki et al., 1989).These approaches, used singly or in combination, have all been appliedas DNA-based methods for tissue-typing of class I and class II HLAspecificities.

[0008] For class I alleles, hypervariable regions are found at differentdegrees in both exon 2 and exon 3, which encode the peptide bindinggroove of the class I molecule. Polymorphism within class II iscontained mainly within defined hypervariable regions in exon 2. Thesepolymorphisms make differentiation between alleles achievable throughhybridization with relevant probes.

AIMS OF THE INVENTION

[0009] It is an aim of the present invention to provide a method fortyping of the alleles HLA-DRB1*0820, HLA-DRB1*04new, HLA-DRB4*01new,HLA-B*3913, HLA-B*1406 and/or HLA-B*51new.

[0010] It is a more specific aim of the present invention to provide amethod for typing of said alleles, with said method comprising anamplification step and a hybridization step.

[0011] It is also an aim of the present invention to provide primers forsaid amplification step.

[0012] It is also an aim of the present invention to provide probes forsaid hybridization step.

[0013] It is also an aim of the present invention to provide adiagnostic kit enabling said method for typing.

[0014] It is another aim of the present invention to provide a methodfor detection of the protein fragments encode by the HLA-DRB1*0820,HLA-DRB1*04new, HLA-DRB4*01new, HLA-B*3913, HLA-B*1406 and/orHLA-B*51new genes.

[0015] It is another aim of the present invention to provide anantiserum or a ligand for use in the detection of said proteinfragments.

[0016] It is another aim of the present invention to provide adiagnostic kit for the detection of said protein fragment.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention discloses the sequence of exon 2 of the HLAallele DRB1*0820. This sequence is identified by SEQ ID NO 1 and isshown below.5                  10                  15                  20 (47) CACGT TTC TTG GAG TAC TCT ACG TCT GAG TGT CAT TTC TTC AAT GGG (SEQ IDNO 1)                    25                  30                  35 (92)   ACG GAG CGG GTG CGG TTC CTG GAC AGA TAC TTC TAT AAC CAA GAG                   40                  45                  50 (137)   GAG TAC GTG CGC TTC GAC AGC GAG GTG GGG GAG TAG CGG GCG GTG                   55                  60                  65 (182)   ACG GAG CTG GGG CGG CCT GAT GCC GAG TAG TGG AAC AGC CAG AAG                   70                  75                  80 (227)   GAG TTC GTG GAA GAG AGG CGG GCC CTG GTG GAC ACC TAC TGC AGA                   85                  90    GAG AAG TAC GGG GTT GTG GAGAGC TTC AGA GTG GAG GGG CGA

[0018] The sequence is shown from 5′ to 3′ and runs from codon 5 tocodon 94 of exon 2. The numbering of the codons is indicated. Thenucleotide positions are indicated between brackets. This sequence hasbeen submitted to the EMBL database and was assigned the accessionnumber AJ000927. The allele DRB1*0820 is a novel allele that has notbeen previously described.

[0019] The present invention also discloses the sequence of exon 2 ofthe HLA allele DRB1*04new. This sequence is identified by SEQ ID NO 50and is shown below.  6             10                  15                  20 (43)   T TTCTTG GAG CAG GTT AAA CCT GAG TGT CAT TTC TTC AAC GGG (SEQ ID NO 50)                25                  30                  35 (88) ACG GAGCGG GTG GGG TTC CTG GAC AGA TAG TTC TAT CAC CAA GAG                40                  45                  50 (133) GAG TACGTG CGC TTG GAC AGC GAC GTG GGG GAG TAG CGG GCG GTG                55                  60                  65 (178) ACG GAGCTG GGG CGG CCT GAT GCC GAG TAC TGG AAC AGC GAG AAG                70                  75                  80 (223) GAG CTGGTG GAG GAG AAG GGG CCC GCG GTG GAG ACC TAC TGG AGA                 85GAG AAG TAG GGG GTT GGT GA

[0020] The sequence is shown from 5′ to 3′ and runs from codon 6 tocodon 87 of exon 2. The numbering of the codons is indicated. Thenucleotide positions are indicated between brackets.

[0021] This sequence has been submitted to the EMBL database and wasassigned the accession number AJ133492. The allele DRB1*04new is a novelallele that has not been previously described.

[0022] The present invention also discloses the sequence of exon 2 ofthe HLA allele DRB4*01new. This sequence is identified by SEQ ID NO 67and is shown below.5                  10                  15                  20 (47) CAGGT TTG TTG GAG CAG GCT AAG TGT GAG TGT CAT TTG GTG AAT GGG (SEQ ID NO67)                    25                  30                  35 (92)   ACG GAG GGA GTG TGG AAC CTG ATC AGA TAC ATC TAT AAC CAA GAG                   40                  45                  50 (137)   GAG TAC GGG GGG TAC AAC AGT GAT CTG GGG GAG TAG GAG GGG GTG                   55                  60                  65 (182)   AGG GAG GTG GGG GGG CCT GAC GCT GAG TAG TGG AAC AGG GAG AAG                   70                  75                  80 (227)   GAG CTG CTG GAG CGG AGG CGG GCC GAG GTG GAG ACC TAG TGC AGA                   85                  90                  95 (270)   TAG AAG TAG GGG GTT GTG GAG AGC TTC AGA GTG GAG CGG GGA G

[0023] The sequence is shown from 5′ to 3′ and runs from codon 5 tocodon 95 of exon 2. The numbering of the codons is indicated. Thenucleotide positions are indicated between brackets. This sequence hasbeen submitted to the EMBL database and was assigned the accessionnumber AJ131789. The allele DRB4*01new is a novel allele that has notbeen previously described.

[0024] Having knowledge of this sequence information, the skilled manwill be able to devise methods that enable typing of said allele. Thepresent invention thus relates to a method for typing of the allelesHLA-DRB1*0820, HLA-DRB1*04new and/or HLA-DRB4*01new in a sample.

[0025] According to a preferred embodiment, the present inventionrelates to a method for typing of the alleles HLA-DRB1*0820,HLA-DRB1*04new and/or HLA-DRB4*01new in a sample, with said methodcomprising:

[0026] i) amplifying a fragment comprising all or part of exon 2 of saidallele using at least one suitable pair of primers,

[0027] ii) hybridizing the amplified product of step i) to a set ofprobes, with the probes of said set specifically hybridizing to targetregions comprising one or more polymorphic nucleotides in exon 2 of saidallele,

[0028] iii) determining from the result of step ii) the presence orabsence of the alleles HLA-DRB1*0820, HLA-DRB1*04new and/orHLA-DRB4*01new in the sample.

[0029] The primers used in this method may be generic primers, i.e.primers that hybridize to target regions that are conserved, at leasttowards their 3′-end, amongst all members of a group of alleles (e.g.the DPB group or the DQB group or the DRB group) and thus will lead toamplification of all alleles within this group. Alternatively theprimers may be subgroup-specific, i.e. primers that hybridize to targetsequences that are only present in a subgroup of alleles. Suchsubgroup-specific primers can be used separately, or more than one5′-primer or more than one 3′-end primer can be used together in a mix.Such a mix is sometimes called a multiplex primer. Different types ofprimers may be used in combination, e.g. a multiplex 5′-primer may beused with a generic 3′-primer, etc.

[0030] According to a more preferred embodiment, the present inventionrelates to a method as defined above, further characterized in that thepimers used for the amplification of exon 2 of DRB1*0820, HLA-DRB1*04newand/or HLA-DRB4*01new are chosen from Table 1. TABLE 1 Primers foramplification of exon2 of the HLA-allele DRB1*0820, the HLA-alleleDRB1*04new and/or the HLA-allele DRB4*01new. Primer Position¹ andsequence SEQ ID NO DRBp5′ gen intron-GATCCTTCGTGTCCCCACAGCACG-6 SEQ IDNO 3 tl,43 DRBp5′ intron intron-ACCGGATCCTTCGTGTCCCCACAG-5 SEQ ID NO 53DRBp5′ DR3, 8, intron-CCCCACAGCACGTTTCTTGGAGTACTC-11 SEQ ID NO 4 11, 12,13, 14 DRBp5′ DR1, 7 intron-TGTCCCCACAG CA CGT TTC TTG TG-9 SEQ ID NO 5DRBp5′ DR4 6-T TTC TTG GAG GAG GTT AAA C-13 SEQ ID NO 6 DRBp5′ DR4 5-ACGT TTG TTG GAG GAG GTT AAA C-13 SEQ ID NO 52 DRBp5′ DR9 5-CA CGT TTCTTG AAG GAG GAT AAG TT-13 SEQ ID NO 7 DRBp5′ DR10 intron-CACAG CA CGTTTC TTG GAG G-10 SEQ ID NO 8 DRBp5′ DR15, 16 8-CTG TGG GAG CCT AAGAGG-13 SEQ ID NO 9 DRB4p5′ 9-TAAGTGTGAGTCTGATTTG-17 SEQ ID NO 54DRBp3′ gen 94-TCGCCGCTGCACTGTGAAGCTC-87 SEQ ID NO 10 DRBp3′ DRB1intron-ATTGGGGGGCGGCGCT-intron SEQ ID NO 11 DRB3′ cod86V92-CTGCACTGTCAAGCTGTCCA-86 SEQ ID NO 12 DRBp3′ intronintron-CCGGGGGTCCACCATGCTCAC-95 SEQ ID NO 107

[0031] SEQ ID NO 3 and SEQ ID NO 53 are generic primers that have atarget region covering the junction of intron 1 and exon 2. This targetregion is conserved amongst all DRB alleles (DRB1, DRB3, DRB4 and DRB5).SEQ ID NOs 4 to 9 and SEQ ID NOs 4, 5, 52, 7, 8, 9 constitute multiplexprimer mixes, which hybridize to the subclasses DR1, DR3, DR4, DR7, DR8,DR9, DR10, DR11, DR12, DR13, DR14, DR15 and DR16. Together thesesubclasses constitute the group of DRB1 alleles. SEQ ID NO 4 is the onlymember of these primer mixes that specifically hybridizes to the alleleHLA-DRB1 *0820. SEQ ID NOs 6 and 52 are the only members of the primermixes that specifically hybridize to the allele HLA-DRB1*04new. SEQ IDNO 54 specifically hybridizes to the allele HLA-DRB4. These specificprimers can be used separately or in a mix such as described above. SEQID NO 10 is a generic primer that has its target region at codon 94 tocodon 87 in exon 2. This target region is conserved amongst all DRBalleles. SEQ ID NO 11 is situated in intron 2, and hybridizes to all DRB1 alleles. The target region of SEQ ID NO 12 is situated at codon 92 tocodon 86. Codon 86 is a dimorphic codon that either encodes a Val or aGly. SEQ ID NO 12 hybrizes to the codon encoding Val. SEQ ID NO 107 issituated in intron 2 and hybridizes to all DRB alleles.

[0032] According to another more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said polymorphic nucleotides have the following positions in SEQ IDNO 1 and SEQ ID NO 67:

[0033] 9, 12, 13, 15, 16, 17, 18, 19, 22, 23, 24, 25, 26, 27, 29, 33,35, 44, 61, 64, 65, 67, 69, 71, 75, 76, 78, 81, 84, 89, 92, 95, 96, 97,99, 100, 104, 106, 127, 136, 146, 156, 157, 158, 160, 161, 162; 165,166, 186, 194, 195, 196, 197, 198, 199, 203, 205, 207, 208, 209, 217,218, 219, 220, 221, 237, 239, 241, 244, 245, 251, 257; and

[0034] the following positions in SEQ ID NO 50:

[0035]5, 8, 9, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 25, 29, 31,40, 57, 60, 61, 63, 65, 67, 71, 72, 74, 77, 80, 85, 88, 91, 92, 93, 95,96, 100, 102, 123, 132, 142, 152, 153, 154, 156, 157, 158, 161, 162,182, 190, 191, 192, 193, 194, 195, 199, 201, 203, 204, 205, 213, 214,215, 216, 217, 233, 235, 237, 240, 241.

[0036] These nucleotides are shown in boldface in the sequences above(SEQ ID NOs 1, 50 and 67).

[0037] According to an even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 of the alleleDRB1*0820, are chosen from Table 2. TABLE 2 Oligonucleotide probes thatcan be used for typing of the HLA-allele DRB1*0820. Ref. No Position¹and sequence SEQ ID NO 9 9-G TAC TCT ACG TCT GAG TG-15 SEQ ID NO 13 2654-G CCT GAT GCC GAG TAC TGG-61 SEQ ID NO 14 25 64-AG AAG GAC TTC CTGGAA GA-70 SEQ ID NO 15 21 69-A GCC AGG CGG GCC CTG GTG GA- SEQ ID NO 1676 44 84-GGG GTT GTG GAG AGC-88 SEQ ID NO 17 17-TTC TTC AAT GGG ACGGAG-22 SEQ ID NO 18 26-TTC CTG GAC AGA TAG TTC-31 SEQ ID NO 19 34-CAAGAG GAG TAG GTG CGC-39 SEQ ID NO 20 45-GGG GAG TAG CGG GCG GTG-50 SEQ IDNO 21

[0038] According to another even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 of the alleleDRB1*04new, are chosen from Table 3. TABLE 3 Oligonucleotide probes thatcan be used for typing of the HLA-allele DRB1*04new Ref. No Position¹and sequence SEQ ID NO 15 70-CAG AAG CGG GCC GCG-74 SEQ ID NO 55 2432-AT CAC CAA GAG GAG TAC GTG-38 SEQ ID NO 56 27 81-CAC AAC TAC GGG GTTGGT GA-87 SEQ ID NO 57 36 55-G CCT GAT GCC GAG TAC TGG-61 SEQ ID NO 5849 73-GCC GCG GTG GAC AGC-77 SEQ ID NO 59 63 63-C CAG AAG GAC CTC CTGGA-69 SEQ ID NO 60 73 28-C AGA TAC TTC TAT CAC CAA GA- SEQ ID NO 61 35

[0039] According to another even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 of the alleleDRB4*01new, are chosen from Table 4. TABLE 4 Oligonucleotide probes thatcan be used for typing of the HLA-allele DRB4*04new. Ref. No Position¹and sequence SEQ ID NO 15 72-GG GCC GAG GTG GAC A-77 SEQ ID NO 62 3663-C CAG AAG GAC CTC CTG GA-69 SEQ ID NO 63 44 84-GGG GTT GTG GAG AGC-88SEQ ID NO 64 60 35-GAG GAG TAC GCG CGC T-40 SEQ ID NO 65 62 22-GAG CGAGTG TGG AAC C-27 SEQ ID NO 66

[0040] These probes hybridize to target regions comprising polymorphicnucleotides in exon 2. The set of probes with SEQ ID NOs 13 to SEQ ID NO17 yield a unique hybridization pattern for the allele DRB1*0820, thatallows discrimination of this allele from all other DRB1 alleles at theallelic level. The set of probes with SEQ ID NO 55 to SEQ ID NO 61 yielda unique hybridization pattern for the allele DRB1*04new, that allowsdiscrimination of this allele from all other DRB1 alleles at the alleliclevel. The set of probes with SEQ ID NO 62 to SEQ ID NO 66 yield aunique hybridization pattern for the allele DRB4*01new, that allowsdiscrimination of this allele from all other DRB4 alleles at the alleliclevel. At present, 215 different DRB1 alleles and 8 different DRB4alleles have been described (http://www.ebi.ac.uk/imgt/hla/). The probeswith SEQ ID NOs 13 to 17 and 55 to 66 are part of the DRB decoder kit(2nd generation) of Innogenetics NV (Ghent, Belgium), which comprises 62probes. SEQ ID NOs 13 to 17 constitute a subgroup of probes of this kitthat specifically hybridize to the allele DRB1*0820. SEQ ID NOs 55 to 61constitute a subgroup of probes of this kit that specifically hybridizeto the allele DRB1*04new. SEQ ID NOs 62 to 66 constitute a subgroup ofprobes of this kit that specifically hybridize to the allele DRB4*01new.The probes with SEQ ID NOs 13 to 17 and 55 to 66 have been optimized tofunction in combination at the same conditions in a LiPA assay (seebelow).

[0041] The skilled man will recognize that the probes and primers withSEQ ID NOs 3 to 21 and 52 to 66 may be adapted by addition or deletionof one or more nucleotides at their extremities. Such adaptations may berequired, for instance, if the conditions of amplification orhybridization are changed, or if the amplified material is RNA insteadof DNA, as is the case in the NASBA system. Different techniques can beapplied to perform the methods of the present invention. Thesetechniques may comprise immobilizing the HLA polynucleic acids, possiblyafter amplification, on a solid support and performing hybridizationwith labelled oligonucleotide probes. Alternatively, the probes may beimmobilized on a solid support and hybridization may be performed withlabelled HLA polynucleic acids, possibly after amplification. Thistechnique is called reverse hybridization. A convenient reversehybridization technique is the line probe assay (LiPA). This assay usesoligonucleotide probes immobilized as parallel lines on a solid supportstrip (Stuyver et al., 1993). It is to be understood that any othertechnique for detection of the above-mentioned HLA allele is alsocovered by the present invention.

[0042] The present invention also relates to any primer or any probe asindicated above, for use in a method for typing of the allelesHLA-DRB1*0820, HLA DRB1*04new and/or HLA-DRB4*01new. The inventionfurther relates to an isolated polynucleic acid, defined by SEQ ID NOs1, 50 and 67, corresponding to exon 2 of the allele HLA-DRB1 *0820, HLADRB1*04new and HLA-DRB4*01new, respectively. The invention also relatesto any fragment thereof that can be used as a primer or as a probe in amethod for typing of said allele.

[0043] Furthermore, having access to the isolated polynucleic acidsdefined by SEQ ID NO 1, a man skilled in the art will be able to isolatethe complete HLA-DRB1*0820 gene from a human genomic library. Havingaccess to the isolated polynucleic acid defined by SEQ ID NO 50, a manskilled in the art will be able to isolate the complete HLA-DRB1*04newgene from a human genomic library. Having access to the isolatedpolynucleic acid defined by SEQ ID NO 67, a man skilled in the art willbe able to isolate the complete HLA-DRB4*01new gene from a human genomiclibrary. This can be done by screening of the library with thepolynucleic acid defined by respectively SEQ ID NO 1, SEQ ID NO 50 orSEQ ID NO 67 or suitable fragments thereof as a hybridisation probe. Thepresent invention thus also relates to the complete HLA-DRB1*0820 gene,the complete HLA-DRB1*04new gene and the complete HLA-DRB4*01new gene.

[0044] According to another preferred embodiment, the present inventionrelates to a diagnostic kit enabling typing of the allelesHLA-DRB1*0820, HLA-DRB1*04new and/or HLA-DRB4*01new, with said kitcomprising at least one primer and/or at least one probe as indicatedabove. Optionally, this kit may also comprise an enzyme and/or reagentsenabling the amplification step and/or reagents enabling thehybridization step.

[0045] According to another preferred embodiment, the present inventionrelates to the protein fragment that is encoded by SEQ ID NO 1. Thesequence of this fragment can be obtained by converting the nucleic acidsequence of SEQ ID NO 1 into the corresponding amino acid sequence,whereby the reading frame to be used is as indicated above. The aminoacid sequence is shown below as SEQ ID NO 2. (SEQ ID NO 2)   R F L E Y ST S E C H F F N G T E R V R F L D R Y F Y N Q E E Y V R F D S D V G E YR A V T E L G R P D A E Y W N S Q K D F L E D R R A L V D T Y C R H N YG V V E S F T V Q R R

[0046] According to another preferred embodiment, the present inventionrelates to the protein fragment that is encoded by SEQ ID NO 50. Thesequence of this fragment can be obtained by converting the nucleic acidsequence of SEQ ID NO 50 into the corresponding amino acid sequence,whereby the reading frame to be used is as indicated above. The aminoacid sequence is shown below as SEQ ID NO 51. (SEQ ID NO 51)     F L E QV K P E C H F F N G T E R V R F L D R Y F Y H Q E E Y V R F D S D V G EY R A V T E L G R P D A E Y W N S Q K D L L E Q K R A A V D T Y C R H NY G V G

[0047] According to another preferred embodiment, the present inventionthus also relates to a method for detection of the protein fragmentsidentified as SEQ ID NO 2 and/or SEQ ID NO 51 in a sample. Said methodmay be one of the well-known serological methods mentioned above(Terasaki and McClelland, 1964, Kissmeyer et al., 1969).

[0048] In accordance the present invention also relates to an antiserumor a ligand binding to the protein fragment according to the invention.The term “a ligand” refers to any molecule able to bind the proteinfragment of the present invention. The latter term specifically refersto polyclonal and/or monoclonal antibodies specifically raised (by anymethod known in the art) against the protein fragment of the presentinvention and also encompasses any antibody-like, and other, constructsas described in detail in WO 98/58965 to Lorré et al.

[0049] The present invention further relates to a kit for the detectionof one or more of the protein fragments of the invention, comprising atleast an antiserum or a ligand as described above.

[0050] The present invention also discloses the sequence of exon 2 andof exon 3 of the HLA allele B*3913. These sequences are identified bySEQ ID NOs 22 and 23 and are shown below.         10         20         30         40         50         60GCTCCCACTC CATGAGGTAT TTCTACACCT CCGTGTCCCG GCCCGGCCGC GGGGAGCCCG (SEQID NO 22)         70         80         90         100        110        120GCTTCATCTC AGTGGGCTAC GTGGACGACA CGCAGTTCGT GAGGTTCGAC AGCGACGCCG         130        140        150        160        170        180CGAGTCCGAG AGAGGAGCCG CGGGCGCCGT GGATAGAGCA GGAGGGGCCG GAGTATTGGG         190        200        210        220        230        240ACCGGGAGAC ACAGATCTCC AAGACCAACA CACAGACTTA CCGAGAGAGC CTGCGGAACC         250        260        270 TGCGCGGCTA GTACAACCAGAGCGAGGCCG            exon 2         10         20         30         40         50         60GGTCTCACAC CCTCCAGAGG ATGTACGGCT GCGACGTGGG GCCGGACGGG CGCCTCCTCC (SEQID NO 23)         70         80         90         100        110        120GCGGGCATAA CCAGTTCGCC TACGACGGCA AGGATTACAT CGCCCTGAAC GAGGACCTGA         130        140        150        160        170        180GCTCCTGGAC CGCGGCGGAC ACCGCGGCTC AGATCACCCA GCGCAAGTGG GAGGCGGCCC         190        200        210        220        230        240GTGTGGCGGA GCAGCTGAGA ACCTACCTGG AGGGCACGTG CGTGGAGTGG CTCCGCAGAT         250        260        270 ACCTGGAGAA CGGGAAGGAG ACGCTGCAGCGCGCGG     exon 3

[0051] These sequences are shown from 5′ to 3′. These sequences havebeen submitted to the EMBL database and were assigned the accessionnumber AJ223282. The allele HLA-B*3913 is a novel allele that has notbeen previously described.

[0052] The present invention also discloses the sequence of exon 2 andof exon 3 of the HLA allele B*1406. These sequences are identified bySEQ ID NOs 72 and 73 and are shown below.         10         20         30         40         50         60GCTCCCACTC CATGAGGTAT TTCTACACCG CCGTGTCCCG GCCCGGCCGC GGGGAGCCCC (SEQID NO 72)         70         80         90         100        110        120GCTTCATCTC AGTGGGCTAC GTGGACGACA CGCAGTTCGT GAGGTTCGAC AGCGACGCCG         130        140        150        160        170        180CGAGTCCGAG AGAGGAGCCG CGGGCGCCGT GGATAGAGCA GGAGGGGCCG GAATATTGGG         190        200        210        220        230        240ACCGGAACAC ACAGATCTGC AAGACCAACA CACAGACTGA CCGAGAGAGC CTGCGGAACC         250        260        270 TGCGCGGCTA CTACAACCAGAGCGAGGCCG            exon 2         10         20         30         40         50         60GGTCTCACAC CCTCCAGAGG ATGTACGGCT GCGACGTGGG GCCGGACGGG CGCCTCCTCC (SEQID NO 73)         70         80         90         100        110        120GCGGGTATAA CCAGTTCGCC TACGACGGCA AGGATTACAT CGCCCTGAAC GAGGACCTGA         130        140        150        160        170        180GCTCCTGGAC CGCGGCGGAC ACCGCGGCTC AGATCACCCA GCGCAAGTGG GAGGCGGCCC         190        200        210        220        230        240GTGAGGCGGA GCAGCTGAGA GCCTACCTGG AGGGCACGTG CGTGGAGTGG CTCCGCAGAC         250        260        270 ACCTGGAGAA CGGGAAGGAG ACGCTGCAGCGCGCGG     exon 3

[0053] These sequences are shown from 5′ to 3′. These sequences havebeen submitted to the EMBL database and were assigned the accessionnumbers AJ131193 for exon 2 and AJ131194 for exon 3. The alleleHLA-B*1406 is a novel allele that has not been previously described.

[0054] The present invention also discloses the sequence of exon 2 andof exon 3 of the HLA allele B*51new. These sequences are identified bySEQ ID NOs 74 and 75 and are shown below.         10         20         30         40         50         60GCTCCCACTC CATGAGGTAT TTCTACACCG CCATGTCCCG GCCCGGCCGC GGGGAGCCCC (SEQID NO 74)         70         80         90         100        110        120GCTTCATTGC AGTGGGCTAC GTGGACGACA CCCAGTTCGT GAGGTTCGAC AGCGACGCCG         130        140        150        160        170        180CGAGTCCGAG GACGGAGCCC CGGGCGCCAT GGATAGAGCA GGAGGGGCCG GAGTATTGGG         190        200        210        220        230        240ACCGGAACAC ACAGATCTTC AAGACCAACA CACAGACTTA CCGAGAGAAC CTGCGGATCG         250        260        270 CGCTCCGCTA CTACAACCAGAGCGAGGCCG            exon 2         10         20         30         40         50         60GGTCTCACAC TTGGCAGACG ATGTATGGCT GCGACGTGGG GCCGGACGGG CGCCTCCTCC (SEQID NO 75)         70         80         90         100        110        120CCGGGCATAA CCAGTACGCC TACGACGGCA AAGATTACAT CGCCCTGAAC GAGGACCTGA         130        140        150        160        170        180GCTCCTGGAC CGCGGCGGAC ACCGCGGCTC AGATCACCCA GCGCAAGTGG GAGGCGGCCC         190        200        210        220        230        240GTGAGGCGGA GCAGCTGAGA GCCTACCTGG AGGGCCTGTG CGTGGAGTGG CTCCGCAGAC         250        260        270 ACCTGGAGAA CGGGAAGGAG TCGCTGCAGCGCGCGG     exon3

[0055] These sequences are shown from 5′ to 3′. The allele HLA-B*51newis a novel allele that has not been previously described.

[0056] Having knowledge of this sequence information, the skilled manwill be able to devise methods that enable typing of said alleles. Thepresent invention thus relates to a method for typing of the allelesHLA-B*3913, HLA-B*1406 and/or HLA-B*51new in a sample.

[0057] According to a preferred embodiment, the present inventionrelates to a method for typing of the alleles HLA-B*3913, HLA-B*1406and/or HLA-B*51new in a sample, with said method comprising:

[0058] i) amplifying a fragment of said allele comprising all or part ofexon 2 and/or all or part of exon 3 of said allele using at least onesuitable pair of primers;

[0059] ii) hybridizing the amplification product of step i) to at leastone probe that specifically hybridizes to a target region comprising oneor more polymorphic nucleotides in exon 2 or in exon 3 of said allele;

[0060] iii) determining from the result of step ii) the presence orabsence of the allele HLA-B*3913, HLA-B*1406 and/or HLA-B*51new in thesample.

[0061] The primers used in this method may be generic primers, i.e.primers that hybridize to target regions that are conserved, at leasttowards their 3′-end, amongst all alleles of a given locus (e.g. theHLA-A alleles or the HLA-B alleles or the HLA-C alleles) and thus willlead to amplification of all alleles of this locus. Alternatively theprimers may be subgroup-specific, i.e. primers that hybridize to targetsequences that are only present in a subgroup of alleles. Thesesubgroup-specific primers can be used separately, or more than one5′-primer or more than one 3′-end primer can be used together in a mix.Such a mix is sometimes called a multiplex primer. Different types ofprimers may be used in combination, e.g. a multiplex 5′-primer may beused with a generic 3′-primer.

[0062] According to a more preferred embodiment, the present inventionrelates to a method as defined above, further characterized in that theprimers used are chosen from Table 5. TABLE 5 Primers for amplificationof exon2/exon 3 of the HLA-allele B*3913, the HLA-allele B*1406 and/orthe HLA-allele B*51new. Name Sequence SEQ ID NO IBPIN1GGGAGGAGCGAGGGGACCSCAC SEQ ID NO 26 (S = G OR C) IBPIN3GGAGGCCATCCCCGGCGACCTAT SEQ ID NO 27

[0063] IBPIN1 is a 5′-primer, located upstream of exon 2 and IBPIN3 is a3′-primer, located downstream of exon 3. These are generic primers,enabling amplification of a fragment of all 274 HLA-B alleles that arepresently known (http://www.ebi.ac.uk/imgt/hla/).

[0064] According to another more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat:

[0065] said polymorphic nucleotides have the following positions in exon2:

[0066] 11, 24, 30, 33, 44, 46, 68, 69, 71, 88, 92, 94, 102, 120, 131,132, 133, 136, 140, 149, 153, 155, 161, 173, 174, 183, 186, 188, 190,193, 196, 197, 198, 199, 200, 204, 205, 207, 208, 209, 210, 212, 219,226, 228, 229, 236, 238, 240, 241, 244, 246, 268, and/or

[0067] said polymorphic nucleotides have the following positions in exon3:

[0068] 2, 10, 11, 12, 13, 14, 18, 19, 20, 26, 36, 44, 54, 66, 68, 69,75, 76, 77, 92, 120, 134, 141, 142, 145, 156, 159, 163, 169, 184, 195,196, 197, 201, 214, 216, 217, 227, 228, 229, 240.

[0069] These polymorphic nucleotides are shown in boldface in thesequences above (SEQ ID NOs 22 and 23, SEQ ID NOs 72 and 73, and SEQ IDNOs 74 and 75).

[0070] According to another even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 or exon 3 ofthe allele HLA-B*3913, are chosen from Table 6. TABLE 6 Oligonucleotideprobes that can be used for typing of the HLA-allele B *3913. ReferenceSequence¹ SEQ ID NO 56 GGGACACGGAGGTGTAGA SEQ ID NO 28 92CCGGCCCGGCCGCGGG SEQ ID NO 29 2 GCTTCATCTCAGTGGGCT SEQ ID NO 30 HCGTTCGTGAGGTTCGACA SEQ ID NO 31 7 GAGTCCGAGAGAGGAGCCG SEQ ID NO 32 87GGGCGGAGTATTGGGAC SEQ ID NO 33 10 GGACCGGGAGACACAGAT SEQ ID NO 34 13AGATCTCCAAGACCAAC SEQ ID NO 35 18 CACAGACTTACCGAGAG SEQ ID NO 36 19ACGGAGAGAGCCTGCGG SEQ ID NO 37 50 CGGAACCTGCGCGGCTA SEQ ID NO 38 26AGAGGATGTACGGCTGC SEQ ID NO 39 GACGTGGGGCCGGACG SEQ ID NO 40 91GACGGGCGCCTCCTCCG SEQ ID NO 41 28 TCCTCCGCGGGCATAACCAG SEQ ID NO 42 53GGGCATAACCAGTTCGCCT SEQ ID NO 43 90 GAGGACCTGAGCTCCTGG SEQ ID NO 44 38CGGCCCGTGTGGCGGAG SEQ ID NO 45 88 GCAGCTGAGAACCTACCT SEQ ID NO 46 36TGGAGGGCACGTGCGTG SEQ ID NO 47 CGTGGAGTGGCTCCGC SEQ ID NO 48TCCGCAGATACCTGGAGA SEQ ID NO 49

[0071] These probes hybridize to target regions comprising polymorphicnucleotides in exon 2 or exon 3 of the allele B*3913. All probes aresense probes, i.e. hybridizing to the anti-sense strand, except theprobe with SEQ ID NO 28, which is an anti-sense probe. The probes withSEQ ID NOs 28 to 49 have been optimized to function under the sameconditions in a LiPA assay (see below).

[0072] According to another even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 or exon 3 ofthe allele HLA-B*1406, are chosen from Table 7. TABLE 7 Oligonucleotideprobes that can be used for typing of the HLA-allele B*1406. ReferenceSequence¹ SEQ ID NO B75 TCTACACCGCCGTGTCC SEQ ID NO 76 B92CCGGCCCGGCCGCGGG SEQ ID NO 29 B2  GCTTCATCTCAGTGGGCT SEQ ID NO 30 B85GCGACGCCGCGAGTCCGA SEQ ID NO 77 B7  GAGTCCGAGAGAGGAGCCG SEQ ID NO 32 B98GGCCGGAATATTGGGAC SEQ ID NO 78 B9  GGACCGGAACACACAG SEQ ID NO 79 B16ACAGATCTGCAAGACCA SEQ ID NO 80 B17 ACAGACTGACCGAGAG SEQ ID NO 81 B19ACCGAGAGAGCCTGCGG SEQ ID NO 37 B56 GGAACCTGCGCGGCTACTA SEQ ID NO 82 B26AGAGGATGTACGGCTG SEQ ID NO 83 B89 CGACGTGGGGCCGGACG SEQ ID NO 84 B91GACGGGCGCCTCCTCCG SEQ ID NO 41 B63 GGGTATAACCAGTTCGCCT SEQ ID NO 85 B90AGGACCTGAGCTCCTGG SEQ ID NO 86 B31 GCCCGTGAGGCGGAGC SEQ ID NO 87 B66GCAGCTGAGAGCCTACCT SEQ ID NO 88 B36 TGGAGGGCACGTGCGTG SEQ ID NO 47 B88CGTGGAGTGGCTCCGC SEQ ID NO 48 B70 CCGCAGACACCTGGAGA SEQ ID NO 89

[0073] These probes hybridize to target regions comprising polymorphicnucleotides in exon 2 or exon 3 of the allele B*1406. All probes aresense probes, i.e. hybridizing to the anti-sense strand. The probes withSEQ ID NOs 30, 32, 79, 80, 81, 37, 82, 83, 87, 47 and 89 have beenoptimized to function under the same conditions in a LiPA assay (seebelow).

[0074] According to another even more preferred embodiment, the presentinvention relates to a method as defined above, further characterized inthat said probes that specifically hybridize to a target regioncomprising one or more polymorphic nucleotides in exon 2 or exon 3 ofthe allele HLA-B*51new, are chosen from Table 8. TABLE 8 Oligonucleotideprobes that can be used ror typing of the HLA-allele B*51new. ReferenceSequence¹ SEQ ID NO B3  GCTTCATTGCAGTGGGCT SEQ ID NO 90 B6 CGAGTCCGAGGACGGAGCCCCGG SEQ ID NO 91 B9  GGACCGGAACACACAG SEQ ID NO 79B14 CAGATCTTCAAGACCAAC SEQ ID NO 92 B18 CACACAGACTTACCGAGAG SEQ ID NO 93B51 CGAGAGAACCTGCGGATC SEQ ID NO 94 B55 CGGATCGCGCTCGGCTA SEQ ID NO 95B73 TCTACACCGCCATGTCC SEQ ID NO 96 B85 GCGACGCCGCGAGTCCG SEQ ID NO 97B87 GGCCGGAGTATTGGGAC SEQ ID NO 33 B23 ACACTTGGCAGACGATG SEQ ID NO 98B31 GCCCGTGAGGCGGAGC SEQ ID NO 87 B35 GGAGGGCCTGTGCGTG SEQ ID NO 99 B60CATAACCAGTACGCCTACG SEQ ID NO 100 B70 CCGCAGACACCTGGAGA SEQ ID NO 89 B88GTGGAGTGGCTCCGC SEQ ID NO 101 B89 CGACGTGGGGCCGGACG SEQ ID NO 84 B90GAGGACCTGAGCTCCTGG SEQ ID NO 44 B91 GACGGGCGCCTCCTCC SEQ ID NO 102

[0075] These probes hybridize to target regions comprising polymorphicnucleotides in exon 2 or exon 3 of the allele B*51new. All probes aresense probes, i.e. hybridizing to the anti-sense strand, except theprobe with SEQ ID NO 97, which is an anti-sense probe. The probes withSEQ ID NOs 90, 91, 79, 92, 93, 98, 87, 99, 94, 95, 100 and 89 have beenoptimized to function under the same conditions in a LiPA assay (seebelow).

[0076] The skilled man will recognize that the probes and primers withSEQ ID NOs 26 to 49 and SEQ ID NOs 76 to 102 may be adapted by additionor deletion of one or more nucleotides at their extremities. Suchadaptations may be required, for instance, if the conditions ofamplification or hybridization are changed, or if the amplified materialis RNA instead of DNA, as is the case in the NASBA system.

[0077] Different techniques can be applied to perform the methods of thepresent invention. These techniques may comprise immobilizing the HLApolynucleic acids, possibly after amplification, on a solid support andperforming hybridization with labelled oligonucleotide probes.Alternatively, the probes may be immobilized on a solid support andhybrization may be performed with labelled HLA polynucleic acids,possibly after amplification. This technique is called reversehybridization. A convenient reverse hybridization technique is the lineprobe assay (LiPA). This assay uses oligonucleotide probes immobilizedas parallel lines on a solid support strip (Stuyver et al., 1993). It isto be understood that any other technique for detection of theabove-mentioned HLA allele is also covered by the present invention.

[0078] The present invention also relates to any primer or any probe asindicated above, for use in a method for typing of the allelesHLA-B*3913, HLA-B*1406 and/or HLA-B*51new. The invention further relatesto an isolated polynucleic acid, defined by SEQ ID NO 22, correspondingto exon 2 of the allele HLA-B*3913, and to an isolated polynucleic acid,defined by SEQ ID NO 23, corresponding to exon 3 of said allele, or toany fragment of said polynucleic acids that can be used as a primer oras a probe in a method for typing of said allele. The invention alsorelates to an isolated polynucleic acid, defined by SEQ ID NO 72,corresponding to exon 2 of the allele HLA-B*1406, and to an isolatedpolynucleic acid, defined by SEQ ID NO 73, corresponding to exon 3 ofsaid allele, or to any fragment of said polynucleic acids that can beused as a primer or as a probe in a method for typing of said allele.The invention also relates to an isolated polynucleic acid, defined bySEQ ID NO 74, corresponding to exon 2 of the allele HLA-B*51new, and toan isolated polynucleic acid, defined by SEQ ID NO 75, corresponding toexon 3 of said allele, or to any fragment of said polynucleic acids thatcan be used as a primer or as a probe in a method for typing of saidallele.

[0079] Furthermore, having access to the isolated polynucleic acidsdefined by SEQ ID NO 22 and SEQ ID NO 23, a man skilled in the art willbe able to isolate the complete HLA-B*3913 gene from a human genomiclibrary. Having access to the isolated polynucleic acids defined by SEQID NO 72 and SEQ ID NO 73, a man skilled in the art will be able toisolate the complete HLA-B*1406 gene from a human genomic library.Having access to the isolated polynucleic acids defined by SEQ ID NO 74and SEQ ID NO 75, a man skilled in the art will be able to isolate thecomplete HLA-B*51new gene from a human genomic library This can be doneby screening of the library with respectively the polynucleic acidsdefined by SEQ ID NO 22 or 23, the polynucleic acids defined by SEQ IDNO 72 or 73, or the polynucleic acids defined by SEQ ID NO 74 or 75, orby any suitable fragments thereof as a hybridisation probe. The presentinvention thus also relates to the complete HLA-B*3913, HLA-B*1406 andHLA-B*51new gene.

[0080] According to another preferred embodiment, the present inventionrelates to a diagnostic kit enabling typing of the alleles HLA-B*3913,HLA-B1406 and/or HLA-B*51new, with said kit comprising at least oneprimer and/or at least one probe as indicated above. Optionally, thiskit may also comprise an enzyme and/or reagents enabling theamplification step and/or reagents enabling the hybridization step.

[0081] According to another preferred embodiment, the present inventionrelates to the protein fragments that are encoded by SEQ ID NO 22 or SEQID NO 23. The sequence of these fragments can be obtained by convertingthe nucleic acid sequences of SEQ ID NOs 22 or 23 into the correspondingamino acid sequences. The amino acid sequences are shown below as SEQ IDNOs 24 and 25 respectively. (SEQ ID NO 24) GSHSMRYFYT SVSRPGRGEPRFISVGYVDD TQFVRFDSDA ASPREEPRAP WIEQEGPEYW DRETQISKTN TQTYRESLRNLRGYYNQSEA (SEQ ID NO 25) GSHTLQRMYG CDVGPDGRLL RGHNQFAYDG KDYIALNEDLSSWTAADTAA QTTQRKWEAA RVAEQLRTYL EGTCVEWLRR YLENGKETLQ RA

[0082] According to another preferred embodiment, the present inventionrelates to the protein fragments that are encoded by SEQ ID NO 72 or SEQID NO 73. The sequence of these fragments can be obtained by convertingthe nucleic acid sequences of SEQ ID NOs 72 or 73 into the correspondingamino acid sequences. The amino acid sequences are shown below as SEQ IDNOs 103 and 104, respectively. (SEQ ID NO 103) GSHSMRYSYT AVSRPGRGEPRFISVGYVDD TQFVRFDSDA ASPREEPRAP WIEQEGPEYW DRNTQTCKTN TQTDRESLRNLRGYYNQSEA (SEQ ID NO 104) GSHTLQRNYG CDVGPDGRLL RGYNQFAYDG KDYIALNEDLSSWTAADTAA QITQRKWEAA REAEQLRAYL EGTCVEWLRR HLENGKETLQ PA

[0083] According to another preferred embodiment, the present inventionrelates to the protein fragments that are encoded by SEQ ID NO 74 or SEQID NO 75. The sequence of these fragments can be obtained by convertingthe nucleic acid sequences of SEQ ID NOs 74 or 75 into the correspondingamino acid sequences. The amino acid sequences are shown below as SEQ IDNOs 105 and 106 respectively. (SEQ ID NO 105) GSHSMRYFYT ANSRPGRGEPRFIAVGYVDD TQFVRFDSDA ASPRTEPRAP WIEQEGPEYW DRNTQTFKTN TQTYRENLRTALRYYNQSEA (SEQ ID NO 106) GSHTWQTMYG CDVGPDGRLL PGHNQYAYDG KDYIALNEDLSSWTAADTAA QITQRKWEAA REAEQLRAYL EGLGVEWLRR HLENGKESLQ RA

[0084] Accordingly, the present invention also relates to a method fordetection of one or more of the protein fragments identified as SEQ IDNOs 24, 25, 103, 104, 105 and/ or 106 in a sample, Said method may beone of the well-known serological methods mentioned above (Terasaki andMcClelland, 1964, Kissmeyer et al., 1969).

[0085] In accordance the present invention also relates to an antiserumor a ligand binding to a polypeptide according of the invention. Theterm “a ligand” refers to any molecule able to bind the polypeptides ofthe present invention. The latter term specifically refers to polyclonaland/or monoclonal antibodies specifically raised (by any method known inthe art) against the polypeptides of the present invention and alsoencompasses any antibody-like, and other, constructs as described indetail in WO 98/58965 to Lorré et al.

[0086] The present invention further relates to a kit for the detectionof a polypeptide of the invention, comprising at least an antiserum or aligand as described above.

DEFINITIONS

[0087] The following definitions and explanations will permit a betterunderstanding of the present invention.

[0088] The target material in the samples to be analysed may either beDNA or RNA, e.g. genomic DNA, messenger RNA or amplified versionsthereof. These molecules are in this application also termed“polynucleic acids”.

[0089] Well-known extraction and purification procedures are availablefor the isolation of RNA or DNA from a sample (e.g. in Sambrook etal.,1989).

[0090] A “polymorphic nucleotide” refers to a nucleotide of the sequenceof a given HLA allele that differs from at least one of the nucleotidesthat are found at the corresponding position in other HLA alleles of thesame locus.

[0091] The term “typing” of an HLA-allele refers to identification ofthe allele, i.e. detection of the allele and discrimination of theallele from other alleles of the same locus.

[0092] The term “probe” according to the present invention refers to asingle-stranded oligonucleotide which is designed to specificallyhybridize to HLA polynucleic acids. Preferably, the probes of theinvention are about 5 to 50 nucleotides long, more preferably from about10 to 25 nucleotides. Particularly preferred lengths of probes include10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25nucleotides. The nucleotides as used in the present invention may beribonu cleotides, deoxyribonucleotides and modified nucleotides such asinosine or nucleotides containing modified groups which do notessentially alter their hybridization characteristics.

[0093] The term “primer” refers to a single stranded oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.Preferably the primer is about 5-50 nucleotides long. Specific lengthand sequence will depend on the complexity of the required DNA or RNAtargets, as well as on the conditions at which the primer is used, suchas temperature and ionic strength. It is to be understood that theprimers of the present invention may be used as probes and vice versa,provided that the experimental conditions are adapted.

[0094] The expression “suitable primer pair” in this invention refers toa pair of primers allowing specific amplification of a HLA polynucleicacid fragment.

[0095] The term “target region” of a probe or a primer according to thepresent invention is a sequence within the HLA polynucleic acids towhich the probe or the primer is completely complementary or partiallycomplementary (i.e. with some degree of mismatch). It is to beunderstood that the complement of said target sequence is also asuitable target sequence in some cases.

[0096] “Specific hybridization” of a probe to a target region of the HLApolynucleic acids means that said probe forms a duplex with part of thisregion or with the entire region under the experimental conditions used,and that under those conditions said probe does not form a duplex withother regions of the polynucleic acids present in the sample to beanalysed.

[0097] “Specific hybridization” of a primer to a target region of theHLA polynucleic acids means that, during the amplification step, saidprimer forms a duplex with part of this region or with the entire regionunder the experimental conditions used, and that under those conditionssaid primer does not form a duplex with other regions of the polynucleicacids present in the sample to be analysed. It is to be understood that“duplex” as used hereby, means a duplex that will lead to specificamplification.

[0098] “Specific amplification” of a fragment of the HLA polynucleicacids means amplification of the fragment for which the primers weredesigned, and not of any other fragment of the polynucleic acids presentin a sample.

[0099] The fact that amplification primers do not have to match exactlywith the corresponding target sequence in the template to warrant properamplification is amply documented in the literature (Kwok et al., 1990).However, when the primers are not completely complementary to theirtarget sequence, it should be taken into account that the amplifiedfragments will have the sequence of the primers and not of the targetsequence. Primers may be labelled with a label of choice (e.g. biotine).The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990) or amplification bymeans of Qβ replicase (Lomeli et al., 1989) or any other suitable methodto amplify nucleic acid molecules known in the art.

[0100] Probe and primer sequences are represented throughout thespecification as single stranded DNA oligonucleotides from the 5′ to the3′ end. It is obvious to the man skilled in the art that any of thebelow-specified probes can be used as such, or in their complementaryform, or in their RNA form (wherein T is replaced by U).

[0101] The probes according to the invention can be prepared by cloningof recombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by excision of the latter from thecloned plasmids by use of the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

[0102] The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984). As most other variationsor modifications introduced into the original DNA sequences of theinvention these variations will necessitate adaptions with respect tothe conditions under which the oligonucleotide should be used to obtainthe required specificity and sensitivity. However the eventual resultsof hybridization will be essentially the same as those obtained with theunmodified oligonucleotides. The introduction of these modifications maybe advantageous in order to positively influence characteristics such ashybridization kinetics, reversibility of the hybrid-formation,biological stability of the oligonucleotide molecules, etc.

[0103] The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead) or a chip. Prior to application to the membrane orfixation it may be convenient to modify the nucleic acid probe in orderto facilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

[0104] The term “labelled” refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bej et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic(biotin, digoxigenin, etc.).

[0105] The “biological sample” may be for instance blood, mouth swab orany other sample comprising genomic DNA.

[0106] For designing probes with desired characteristics, the followinguseful guidelines known to the person skilled in the art can be applied.

[0107] Because the extent and specificity of hybridization reactionssuch as those described herein are affected by a number of factors,manipulation of one or more of those factors will determine the exactsensitivity and specificity of a particular probe, whether perfectlycomplementary to its target or not. The importance and effect of variousassay conditions are explained further herein.

[0108] The stability of the [probe: target] nucleic acid hybrid shouldbe chosen to be compatible with the assay conditions. This may beaccomplished by avoiding long AT-rich sequences, by terminating thehybrids with G:C base pairs, and by designing the probe with anappropriate Tm. The beginning and end points of the probe should bechosen so that the length and % GC result in a Tm about 2-10° C. higherthan the temperature at which the final assay will be performed. Thebase composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be more stable at highertemperatures.

[0109] Conditions such as ionic strength and incubation temperatureunder which a probe will be used should also be taken into account whendesigning a probe. It is known that the degree of hybridization willincrease as the ionic strength of the reaction mixture increases, andthat the thermal stability of the hybrids will increase with increasingionic strength. On the other hand, chemical reagents, such as formamide,urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase thestringency of hybridization. Destabilization of the hydrogen bonds bysuch reagents can greatly reduce the Tm. In general, optimalhybridization for synthetic oligonucleotide probes of about 10-50 basesin length occurs approximately 5° C. below the melting temperature for agiven duplex. Incubation at temperatures below the optimum may allowmismatched base sequences to hybridize and can therefore result inreduced specificity.

[0110] It is desirable to have probes which hybridize only underconditions of high stringency. Under high stringency conditions onlyhighly complementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. The degree of stringency is chosen such as to maximize thedifference in stability between the hybrid formed with the target andthe non-target nucleic acid.

[0111] Regions in the target DNA or RNA which are known to form stronginternal structures inhibitory to hybridization are less preferred.Likewise, probes with extensive self-complementarity should be avoided.As explained above, hybridization is the association of two singlestrands of complementary nucleic acids to form a hydrogen bonded doublestrand. It is implicit that if one of the two strands is wholly orpartially involved in a hybrid that it will be less able to participatein formation of a new hybrid. There can be intramolecular andintermolecular hybrids formed within the molecules of one type of probeif there is sufficient self complementarity. Such structures can beavoided through careful probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

[0112] Standard hybridization and wash conditions are disclosed in theMaterials & Methods section of the Examples. Other conditions are forinstance 3× SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at50° C. Other solutions (SSPE (Sodium saline phosphate EDTA), TMAC(Tetramethyl ammonium Chloride), etc.) and temperatures can also be usedprovided that the specificity and sensitivity of the probes ismaintained. When needed, slight modifications of the probes in length orin sequence have to be carried out to maintain the specificity andsensitivity required under the given circumstances.

[0113] The term “hybridization buffer” means a buffer allowing ahybridization reaction between the probes and the polynucleic acidspresent in the sample, or the amplified products, under the appropriatestringency conditions.

[0114] The term “wash solution” means a solution enabling washing of thehybrids formed under the appropriate stringency conditions.

FIGURE AND TABLE LEGENDS

[0115]FIG. 1 represents a drawing of the result of a LiPA experimentenabling typing of the HLA allele DRB1*0820. The numbers refer to probesof the DRB decoder 2nd generation kit (Innogenetics NV, Ghent, Belgium).The amplification and hybridization steps were performed as described inexample 2.

[0116]FIG. 2 represents a drawing of the result of a LiPA experimentenabling typing of the HLA allele DRB1*04new. The numbers refer toprobes of the DRB decoder 2nd generation kit (Innogenetics NV, Ghent,Belgium). The amplification and hybridization steps were performed asdescribed in example 4.

[0117]FIG. 3 represents a drawing of the result of a LiPA experimentenabling typing of the HLA allele DRB4*01new. The numbers refer toprobes of the DRB decoder 2nd generation kit (Innogenetics NV, Ghent,Belgium). The amplification and hybridization steps were performed asdescribed in example 6.

[0118]FIG. 4 represents a drawing of the result of a LiPA experimentenabling typing of the allele HLA-B*3913. The numbers refer to probespresent in the LiPA HLA-B kit (Innogenetics NV, Ghent, Belgium). Theexperiment was performed as described in example 8.

[0119]FIG. 5 represents a drawing of the result of a LiPA experimentenabling typing of the allele HLA-B*1406. The numbers refer to probespresent in the LiPA HLA-B kit (Innogenetics NV, Ghent, Belgium). Theexperiment was performed as described in example 10.

[0120]FIG. 6 represents a drawing of the result of a LiPA experimentenabling typing of the allele HLA-B*51new. The numbers refer to probespresent in the LiPA HLA-B kit (Innogenetics NV, Ghent, Belgium). Theexperiment was performed as described in example 12.

[0121] Table 1. Primers used for the amplification of exon 2 of theHLA-allele DRB1*0820, the HLA-allele DRB1*04new and/or the HLA-alleleDRB4*01new. The numbers before and after the sequences indicate thecodons where the 5′-ends and the 3′-ends of the primers are located.“Intron” means that the 5′-end and/or the 3′-end is/are located in anintron.

[0122] Table 2. Oligonucleotide probes that can be used for typing ofthe HLA-allele DRB1 *0820. The first column shows reference numbers forsome of the probes. The sequences are given from 5′ to 3′. The numbersbefore and after the sequences indicate the codons where the 5′-ends andthe 3′-ends of the probes are located.

[0123] Table 3. Oligonucleotide probes that can be used for typing ofthe HLA-allele DRB1*04new. The first column shows reference numbers forthe probes. The sequences are given from 5′ to 3′. The numbers beforeand after the sequences indicate the codons where the 5′-ends and the3′-ends of the probes are located.

[0124] Table 4. Oligonucleotide probes that can be used for typing ofthe HLA-allele DRB4*01new. The first column shows reference numbers forthe probes. The sequences are given from 5′ to 3′. The numbers beforeand after the sequences indicate the codons where the 5′-ends and the3′-ends of the probes are located.

[0125] Table 5. Primers used for the amplification of exon2/exon 3 ofthe HLA-allele B*3913, the HLA-allele B*1406 and/or the HLA-alleleB*51new.

[0126] Table 6. Oligonucleotide probes that can be used for typing ofthe HLA-allele B*3913. The first column shows reference numbers for someof the probes. The sequences are given from 5′ to 3′.

[0127] Table 7. Oligonucleotide probes that can be used for typing ofthe HLA-allele B*1406. The first column shows reference numbers for theprobes. The sequences are given from 5′ to 3′.

[0128] Table 8. Oligonucleotide probes that can be used for typing ofthe HLA-allele B*51new. The first column shows reference numbers for theprobes. The sequences are given from 5′ to 3′.

EXAMPLES Example 1 Sequence Determination of the Allele HLA-DRB1*0820

[0129] The allele DRB1*0820 was present in a blood sample from aCaucasian donor. The sample was collected by Dr. Bart Vandekerckhove ofthe Laboratorium Immunohematologie at the BloedtransfusiecentrumOost-Vlaanderen in Belgium. Polynucleic acids were prepared from theblood sample by use of the QIAamp Blood Kit (Qiagen, Hilden, Germany)according to the manufacturer's protocol. An amplification step wasperformed with the multiplex primer mix consisting of primers with SEQID NOs 4 to 9 as the 5′-primer and DRBP3′gen (SEQ ID NO 10) as the3′-end primer. The PCR reaction cycle was composed of the followingsteps:

[0130] 5 min at 95° C.

[0131] 35 times (30 s at 95° C.; 20 s at 58° C.; 30 s at 72° C.)

[0132] 10 min at 72° C.

[0133] The PCR reaction was carried out in 10 mM Tris.HCl pH 8.3; 50 mMKCl, 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequence, corresponding toexon 2 of the allele DRB1*0820 was obtained: (SEQ ID NO 1)  CA CGT TTCTTG GAG TAC TCT ACG TCT GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGGTTC CTG GAC AGA TAC TTC TAT AAC CAA GAG GAG TAC GTG CGC TTC GAC AGC GACGTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC TGGAAC AGC CAG AAG GAC TTC CTG GAA GAC AGG CGG GCC CTG GTG GAC ACC TAC TGCAGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA

Example 2 Typing of the Allele DRB1*0820

[0134] The following method for typing of the allele DRB1*0820 in asample is based on the LiPA technology (Stuyver et al, 1993). DNA isextracted from a blood sample with the QIAamp Blood Kit, as indicated inexample 1. For the amplification step different primer mixes may beused: either a generic primer pair (such as SEQ ID NO 3 and SEQ ID NO10), or a multiplex primer (such as the mix composed of primers with SEQID NO 4 to SEQ ID NO 9) combined with a generic primer (such as SEQ IDNO 10) or a generic primer combined with a primer encompassing thedimorphic codon 86 (such as SEQ ID NO 3 with SEQ ID NO 12). Theamplification reaction is carried out in 10 mM Tris.HCl pH 8.3, 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). The PCR reaction iscomposed of 1 step of 5 min at 95° C., 35 cycles of three steps (30 s at95° C., 20 s at 58° C., 30 s at 72° C.) and 1 step of 10 min at 72° C.The allele DRB1*0820 can subsequently be typed by a reversehybridization step to a panel of oligonucleotide probes that areimmobilized on a nitro-cellulose strip. A set of probes composed of theprobes with SEQ ID NO 13 to SEQ ID NO 17 is sufficient to enabledifferentiation between the allele DRB1*0820 and any other presentlyknown DRB1 allele at the allelic level. However, in clinical samples twodifferent DRB1 alleles are present, which complicates the analysis andnecessitates the use of a larger number of probes. Typing is evenfurther complicated by the fact that also associated DRB alleles exist(DRB3, DRB4, DRB5), which show extensive sequence homology with the DRB1alleles. In FIG. 1, for instance, an amplification reaction was carriedout with the generic primers DRBp5′gen (SEQ ID NO 3) and DRBp3′gen (SEQID NO 10), under the conditions outlined above. The amplified productwas subjected to a reverse hybridization assay, by use of the DRBdecoder 2nd generation kit (Innogenetics NV, Ghent, Belgium) accordingto the manufacturer's protocol. This kit comprises a panel of 62oligonucleotide probes, including the probes with SEQ ID NOs 13 to 17 ofTable 2. FIG. 1 shows the result of the hybridization assay. The numbersindicate the different probes that are present on the strip. From thisresult it can be determined that the allele DRB1*0820 is present in thesample, in combination with the previously described alleles DRB1*04012and the associated alleles DRB4*01011 and DRB4*0103. (These associatedalleles have identical sequences in exon 2). The amplified nucleic acidfragment of allele DRB1 *0820 hybridizes to the probes (lines) 9, 21,25, 26 and 44 on the strip (corresponding to SEQ ID NOs 13, 16, 15, 14and 17 respectively).

Example 3 Sequence Determination of the Allele HLA-DRB1*04new

[0135] The allele DRB1*04new was present in a blood sample from aCaucasian donor. The sample was collected by Dr. P. Jindra of theHematologicko-onkologicke odd. in Plzen, Czech Republic. Polynucleicacids were prepared from the blood sample by use of the QIAamp Blood Kit(Qiagen, Hilden, Germany) according to the manufacturer's protocol.

[0136] In a first experiment, exon 2 was cloned in the pGEMt-vector(Promega, Madison, Wis., USA) after amplification with primer DRBp5′gen(SEQ ID NO 3) as the 5′-primer and DRBp3′gen (SEQ ID NO 10) as the3′-primer. The PCR reaction cycle was composed of the following steps:

[0137] 5 min at 95° C.

[0138] 35 times (30 s at 95° C., 20 s at 58° C.; 30 s at 72° C.)

[0139] 10 min at 72° C.

[0140] The PCR reaction was carried out in 10 mM Tris.HCl pH 8.3; 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the SP6and T7 primers provide by Eurogentec (Seraing, Belgium). The followingsequence, corresponding to exon 2 of the allele DRB1*04new was obtained:(SEQ ID NO 68) G ATC CTT CGT GTC CCC ACA GCA CGT TTC TTG GAG CAG GTT AAACCT GAG TGT  CAT TTC TTC AAC GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA TACTTC TAT  CAC CAA GAG GAG TAC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGGGCG  GTG ACG GAG CTG GGG CGG CCT GAT GCC GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CAG AAG CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA GTG CAG CGG CGA

[0141] The position of the generic primers used for the amplification ofexon 2 is shown in bold.

[0142] In a second experiment, exon 2 was amplified with primerDRBp5′DR4 (SEQ ID NO 52) as the 5′-primer and DRBp3′gen (SEQ ID NO 10)as the 3′-primer. The PCR reaction cycle was composed of the followingsteps:

[0143] 5 min at 95° C.

[0144] 35 times (30 s at 95° C.; 20 s at 58° C.; 30 s at 72° C.)

[0145] 10 min at 72° C.

[0146] The PCR reaction was carried out in 10 mM Tris.HCl pH 8.3; 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine, 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequence, corresponding toexon 2 of the allele DRB1*04new was obtained: (SEQ ID NO 69) A CGT TTCTTG GAG CAG GTT AAA CCT GAG TGT CAT TTC TTC AAC GGG ACG GAG  CGG GTG CGGTTC CTG GAC AGA TAC TTC TAT CAC CAA GAG GAG TAC GTG CGC  TTC GAC AGC GACGTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG CGG CCT  GAT GCC GAG TAC TGGAAC AGC CAG AAG GAC CTC CTG GAG CAG AAG CGG GCC  GCG GTG GAC ACC TAC TGCAGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA  GTG CAG CGG CGA

[0147] The position of the primers used for the amplification of exon 2is shown in bold.

Example 4 Typing of the Allele DRB1*04new

[0148] The following method for typing of the allele DRB1*04new in asample is based on the LiPA technology (Stuyver et al, 1993). DNA isextracted from a blood sample with the QIAamp Blood Kit, as indicated inexample 3. For the amplification step different primer mixes may beused: either a generic primer pair (such as SEQ ID NO 3 and SEQ ID NO10), or a multiplex primer (such as the mix composed of primers with SEQID NO 4 to SEQ ID NO 9 or the mix composed of primers with SEQ ID NOs 4,5, 52, 7, 8 and 9) combined with a generic primer (such as SEQ ID NO 10)or a generic primer combined with a primer encompassing the dimorphiccodon 86 (such as SEQ ID NO 3 with SEQ ID NO 12). The amplificationreaction is carried out in 10 mM Tris.HCl pH 8.3; 50 mM KCl; 1.5 mMMgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP (finalconcentrations) and 20 pmol of each primer and 1 U AmpliTaq (AppliedBiosystems, Foster City, Calif., USA). The PCR reaction is composed of 1step of 5 min at 95° C., 35 cycles of three steps (30 s at 95° C., 20 sat 58° C., 30 s at 72° C.) and 1 step of 10 min at 72° C. The alleleDRB1*04new can subsequently be typed by a reverse hybridization step toa panel of oligonucleotide probes that are immobilized on anitro-cellulose strip. A set of probes composed of the probes with SEQID NO 55 to SEQ ID NO 61 is sufficient to enable differentiation betweenthe allele DRB1*04new and any other presently known DRB1 allele at theallelic level. However, in clinical samples two different DRB1 allelesare present, which complicates the analysis and necessitates the use ofa larger number of probes. Typing is even further complicated by thefact that also associated DRB alleles exist (DRB3, DRB4, DRB5), whichshow extensive sequence homology with the DRB1 alleles. In FIG. 2, forinstance, an amplification reaction was carried out with the genericprimers DRBp5′gen (SEQ ID NO 3) and DRBp3′gen (SEQ ID NO 10), under theconditions outlined above. The amplified product was subjected to areverse hybridization assay, by use of the DRB decoder 2nd generationkit (Innogenetics NV, Ghent, Belgium) according to the manufacturer'sprotocol. This kit comprises a panel of 62 oligonucleotide probes,including the probes with SEQ ID NOs 55 to 61 of Table 3. FIG. 2 showsthe result of the hybridization assay. The numbers indicate thedifferent probes that are present on the strip. From this result it canbe determined that the allele DRB1*04new (lines 12, 26, 36, 37, 42, 43,56) is present in the sample, in combination with the previouslydescribed alleles DRB1*1104 (lines 2, 9, 25, 30, 33, 44, 56) and two ofthe associated alleles DRB4*01011 (lines 15, 36, 41, 44, 60, 62) andDRB3*0202 (lines 6, 26, 36, 43, 60). The amplified nucleic acid fragmentof allele DRB1*04new hybridizes to the probes (lines) 12, 26, 36, 37,42, 43 and 56 on the strip (corresponding to SEQ ID NOs 55, 58, 60, 61,56, 57 and 59 respectively).

Example 5 Sequence Determination of the Allele HLA-DRB4*01new

[0149] The allele DRB4*01new was present in a blood sample from aCaucasian donor. The sample was collected by Dr. Bohuslava Jilkova ofthe HLA-Laboratory in Hradec Kralove, Czech Republic. Polynucleic acidswere prepared from the blood sample by use of the QIAamp Blood Kit(Qiagen, Hilden, Germany) according to the manufacturer's protocol.

[0150] In a first experiment, exon 2 was cloned in the pGEMt-vector(Promega, Madison, Wis., USA) after amplification with primerDRBp5′intron (SEQ ID NO 53) as the 5′-primer and DRBp3′intron (SEQ ID NO107) as the 3′-primer. The PCR reaction cycle was composed of thefollowing steps:

[0151] 5 min at 95° C.

[0152] 35 times (30 s at 95° C.; 20 s at 58° C.; 30 s at 72° C.)

[0153] 10 min at 72° C.

[0154] The PCR reaction was carried out in 10 mM Tris.HCl pH 8.3; 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the SP6and T7 primers provide by Eurogentec (Seraing, Belgium). The followingsequence, corresponding to exon 2 of the allele DRB4*01new was obtained:AC CGG ATC CTT CGT GTC CCC ACA GCA CGT TTC TTG GAG CAG GCT AAG TGT (SEQID NO 70) GAG TGT CAT TTC CTC AAT GGG ACG GAG CGA GTG TGG AAC CTG ATCAGA TAC ATC TAT AAC CAA GAG GAG TAC GCG CGC TAC AAC AGT GAT CTG GGG GAGTAC CAG GCG GTG ACG GAG CTG GGG GGG CCT GAC GCT GAG TAC TGG AAC AGC CAGAAG GAC CTC CTG GAG CGG AGG CGG GCC GAG GTG GAC ACC TAC TGC AGA TAC AACTAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA GGT GAG CAT GGT GGA GGGCGG G

[0155] The position of the primers used for the amplification of exon 2is shown in bold.

[0156] In a second experiment, exon 2 was amplified with primer DRB4p5′(SEQ ID NO 54) as the 5′-primer and DRBp3′gen (SEQ ID NO 10) as the3′-primer. The PCR reaction cycle was composed of the following steps:

[0157] 5 min at 95° C.

[0158] 35 times (30 s at 95° C.; 20 s at 58° C.; 30 s at 72° C.)

[0159] 10 min at 72° C.

[0160] The PCR reaction was carried out in 10 mM Tris.HCl pH 8.3, 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequence, corresponding toexon 2 of the allele DRB1*04new was obtained: TAA GTG TGA GTG TCA TTTCCT CAA TGG GAC GGA GCG AGT GTG GAA CCT GAT (SEQ ID NO 71) CAG ATA CATCTA TAA CCA AGA GGA GTA CGC GCG CTA CAA CAG TGA TCT GGG GGA GTA CCA GGCGGT GAC GGA GCT GGG GCG GCC TGA CGC TGA GTA CTG GAA CAG CCA GAA GGA CCTCCT GGA GCG GAG GCG GGC CGA GGT GGA CAC CTA CTG CAG ATC AAC TAC GGG GTTGTG GAG AGC TTC ACA GTG CAG CGG CGA

[0161] The position of the primers used for the amplification of exon 2is shown in bold.

Example 6 Typing of the Allele DRB4*01new

[0162] The following method for typing of the allele DRB4*01new in asample is based on the LiPA technology (Stuyver et al, 1993). DNA isextracted from a blood sample with the QIAamp Blood Kit, as indicated inexample 5. For the amplification step different combinations of 5′ and3′ primers can be used: for example, either a generic primer pair (suchas SEQ ID NO 3 and SEQ ID NO 10 or SEQ ID NO 53 and SEQ ID NO 107), or aspecific primer, such as SEQ ID NO 54, possibly in a mix composed ofother 5′ primers used for the amplification of different DRB alleles(e.g. SEQ ID NO 4 to 9) combined with a generic primer (such as SEQ IDNO 10 or 107) or a generic primer combined with a primer encompassingthe dimorphic codon 86 (such as SEQ ID NO 3 with SEQ ID NO 12). Theamplification reaction is carried out in 10 mM Tris.HCl pH 8.3; 50 mMKCl; 1.5 mM MgCl₂; 0.001% (w/v) gelatine; 200 μM dATP, dGTP, dCTP, dTTP(final concentrations) and 20 pmol of each primer and 1 U AmpliTaq(Applied Biosystems, Foster City, Calif., USA). The PCR reaction iscomposed of 1 step of 5 min at 95° C., 35 cycles of three steps (30 s at95° C., 20 s at 58° C., 30 s at 72° C.) and 1 step of 10 min at 72° C.The allele DR-B4*01new can subsequently be typed by a reversehybridization step to a panel of oligonucleotide probes that areimmobilized on a nitro-cellulose strip. A set of probes composed of theprobes with SEQ ID NO 62 to SEQ ID NO 66 is sufficient to enabledifferentiation between the allele DRB4*01new and any other presentlyknown DRB4 allele at the allelic level. Typing is even furthercomplicated by the fact that also DRB 1 alleles and other associatedalleles exist (DRB3, DRB4, DRB5), which show extensive sequence homologywith the DRB4 alleles. This complicates the analysis and necessitatesthe use of a larger number of probes. In FIG. 3, for instance, anamplification reaction was carried out with the generic primersDRBp5′gen (SEQ ID NO 3) and DRBp3′gen (SEQ ID NO 10), under theconditions outlined above. The amplified product was subjected to areverse hybridization assay, by use of the DRB decoder 2nd generationkit (Innogenetics NV, Ghent, Belgium) according to the manufacturer'sprotocol. This kit comprises a panel of 62 oligonucleotide probes,including the probes with SEQ ID NOs 62 to 66 of Table 4. FIG. 3 showsthe result of the hybridization assay. The numbers indicate thedifferent probes that are present on the strip. From this result it canbe determined that the allele DRB4*01new is present in the sample, incombination with the previously described alleles DRB1 *0403 (lines 15,20, 26, 36, 37, 42, 44), DRB1*1301 (lines 9, 16, 23, 26, 28, 30, 44, 56)and DRB3*0206 (lines 6, 26, 36, 43). The amplified nucleic acid fragmentof allele DRB4*01new hybridizes to the probes (lines) 15, 36, 44, 60 and62 on the strip (corresponding to SEQ ID NOs 62, 63, 64, 65 and 66respectively).

Example 7 Sequence Determination of the Allele HLA-B*3913

[0163] The allele B*3913 was present in a blood sample from a BrazilianCaucasian donor. The sample was collected by Dr. M E Moraes of the Labde Immunogenética, HSE/INCA, Rio De Janeiro, Brazil. Polynucleic acidswere prepared from the blood sample by use of the QIAamp Blood Kit(Qiagen, Hilden, Germany) according to the manufacturer's protocol. Anamplification step was performed with primers IBPIN1 and IBPIN3. The PCRreaction consisted of the following steps:

[0164] 1 min at 96° C.

[0165] 5 times (30 s at 95° C.; 50 s at 64° C.; 50 s at 72° C.)

[0166] 5 times (30 s at 95° C.; 50 s at 62° C.; 50 s at 72° C.)

[0167] 10 times (30 s at 95° C.; 50 s at 60° C.; 50 s at 72° C.)

[0168] 15 times (30 s at 95° C.; 50 s at 55° C.; 50 s at 72° C.)

[0169] 10 min at 72° C.

[0170] The amplification reaction was carried out in 50 mM Tris-HCl pH9.2, 16 mM (NH₄)₂SO₄, 200 μM dNTPs, 2.5 U Taq polymerase, 1.5 mM MgCl₂,15 pmole of each primer and 0.1 to 0.5 μg DNA. Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequences, corresponding toexon 2 and exon 3 of the allele B*3913 were obtained:GCTCCCACTCCATGAGGTATTTCTACACCTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGC (SEQ IDNO 22) TTCATCTCAGTGGGCTACGTGGACGACACGCAGTTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGAGAGGAGCCGCGGGCGCCGTGGATAGAGCACGAGGGGCCGGAGTATTGGGACCGGGAGACACAGATCTCCAAGACCAACACACAGACTTACCGAGAGAGCCTGCGGAACCTGCGCGGCTACTACAACCAGAGCGAGGCCG       exon 2GGTCTCACACCCTCCAGAGGATGTACGGCTGCGACGTGGGGCCGGACGGGCGCCTCCTCCGC (SEQ IDNO 23) GGGCATAACCAGTTCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGAGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGTGGCGGAGCAGCTGAGAACCTACCTGGAGGGCACGTGCGTGGAGTCGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCGCGG        exon 3

Example 8 Typing of the Allele B*3913

[0171] The following method, that may be used to type the allele B*3913in a sample, is based on the LiPA technology (Stuyver et al, 1993).Nucleic acids are extracted from a blood sample with the QIAamp BloodKit, as indicated in example 7. For the amplification step the primerpair IBPIN1 (SEQ ID NO 26) and IBPIN3 (SEQ ID NO 27) is used. The PCRreaction is performed under the same conditions as in example 7. Theallele BLA-B*3913 can subsequently be typed by a reverse hybridizationstep to a panel of oligonucleotide probes that are immobilized on anitro-cellulose strip. For instance, FIG. 4 shows the result of areverse hybidization assay according to the LiPA technique. After anamplification step as described above, the amplified nucleic acids werehybridized to a panel of 60 probes by use of the LiPA HLA-B kit(Innogenetics NV, Ghent, Belgium) according to the manufacturer'sprotocol. From the result shown in FIG. 4, it can be derived that thesample contained the allele HLA-B*3913, in combination with the knownallele HLA-B*52012 The numbers in FIG. 4 indicate probes that arepresent on the strip (note that these probes are not the same probes asthose in FIGS. 1-3). The amplified nucleic acid fragment of alleleHLA-B*3913 hybridizes to the following probes (lines) on the strip: 2(SEQ ID NO 30), 7 (SEQ ID NO 32), 10 (SEQ ID NO 34), 13 (SEQ ID NO 35),18 (SEQ ID NO 36), 19 (SEQ ID NO 37), 26 (SEQ ID NO 39), 28 (SEQ ID NO42), 36 (SEQ ID NO 47), 38 (SEQ ID NO 45), 50 (SEQ ID NO 38), 53 (SEQ IDNO 43) and 56 (SEQ ID NO 28).

Example 9 Sequence Determination of the Allele HLA-B*1406

[0172] The allele B*1406 was present in a blood sample from a BelgianCaucasian donor. The sample was collected by Dr. M P Emonds of theBloodtransfusion Center, Leuven, Belgium. Polynucleic acids wereprepared from the blood sample by use of the QIAamp Blood Kit (Qiagen,Hilden, Germany) according to the manufacturer's protocol. Anamplification step was performed with primers IBPIN1 and IBPIN3. The PCRreaction consisted of the following steps:

[0173] 1 min at 96° C.

[0174] 5 times (30 s at 95° C.; 50 s at 64° C.; 50 s at 72° C.)

[0175] 5 times (30 s at 95° C.; 50 s at 62° C.; 50 s at 72° C.)

[0176] 10 times (30 s at 95° C.; 50 s at 60° C.; 50 s at 72° C.)

[0177] 15 times (30 s at 95° C.; 50 s at 55° C.; 50 s at 72° C.)

[0178] 10 min at 72° C.

[0179] The amplification reaction was carried out in 50 mM Tris-HCl pH9.2, 16 mM (NH₄)₂SO₄, 200 μM dNTPs, 2.5 U Taq polymerase, 1.5 mM MgCl₂,15 pmole of each primer and 0.1 to 0.5 μg DNA. Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequences, corresponding toexon 2 and exon 3 of the allele B*1406 were obtained:GCTCCCACTCCATGAGGTATTTCTACACCGCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGC (SEQ IDNO 72) TTCATCTCAGTGGGCTACGTGGACGACACGCAGTTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGAGAGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAATATTGGGACCGGAACACACAGATCTGCAAGACCAACACACAGACTGACCGAGAGAGCCTGCGGAACCTGCGCGGCTACTACAACCAGAGCGAGGCCG        exon 2GGTCTCACACCCTCCAGAGGATGTACGGCTGCGACGTGGGGCCGGACGGGCGCCTCCTCGGC (SEQ IDNO 73) GGGTATAACCAGTTCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGAGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGAGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGACACCTGGAGAACGGGAAGGAGACGCTGCAGCGCGCGG       exon 3

Example 10 Typing of the Allele B*1406

[0180] The following method, that may be used to type the allele B*1406in a sample, is based on the LiPA technology (Stuyver et al, 1993).Nucleic acids are extracted from a blood sample with the QIAamp BloodKit, as indicated in example 9. For the amplification step the primerpair IBPIN1 (SEQ ID NO 26) and IBPIN3 (SEQ ID NO 27) is used. The PCRreaction is performed under the same conditions as in example 9. Theallele HLA-B*1406 can subsequently be typed by a reverse hybridizationstep to a panel of oligonucleotide probes that are immobilized on anitro-cellulose strip. For instance, FIG. 5 shows the result of areverse hybidization assay according to the LiPA technique. After anamplification step as described above, the amplified nucleic acids werehybridized to a panel of 60 probes by use of the LiPA HLA-B kit(Innogenetics NV, Ghent, Belgium) according to the manufacturer'sprotocol. From the result shown in FIG. 5, it can be derived that thesample contained the allele HLA-B*1406. The numbers in FIG. 5 indicateprobes that are present on the strip (note that these probes are not thesame probes as those in FIGS. 1-3). The amplified nucleic acid fragmentof allele HLA-B*1406 hybridizes to the following probes (lines) on thestrip: 2 (SEQ ID NO 30), 7 (SEQ ID NO 32), 9 (SEQ ID NO 79), 16 (SEQ IDNO 80), 17 (SEQ ID NO 81), 19 (SEQ ID NO 37), 26 (SEQ ID NO 83), 31 (SEQID NO 87), 36 (SEQ ID NO 47), 50 (SEQ ID NO 82) and 55 (SEQ ID NO 89).

Example 11 Sequence Determination of the Allele HLA-B*51new

[0181] The allele B*51new was present in a blood sample from a BrazilianCaucasian donor. The sample was collected by Dr. M E Moraes of the Labde Immunogenética, HSE/INCA, Rio De Janeiro, Brazil. Polynucleic acidswere prepared from the blood sample by use of the QIAamp Blood Kit(Qiagen, Hilden, Germany) according to the manufacturer's protocol. Anamplification step was performed with primers IBPIN1 and IBPIN3. The PCRreaction consisted of the following steps:

[0182] 1 min at 96° C.

[0183] 5 times (30 s at 95° C.; 50 s at 64° C.; 50 s at 72° C.)

[0184] 5 times (30 s at 95° C.; 50 s at 62° C.; 50 s at 72° C.)

[0185] 10 times (30 s at 95° C.; 50 s at 60° C.; 50 s at 72° C.)

[0186] 15 times (30 s at 95° C.; 50 s at 55° C.; 50 s at 72° C.)

[0187] 10 min at 72° C.

[0188] The amplification reaction was carried out in 50 mM Tris-HCl pH9.2, 16 mM (NH₄)₂SO₄, 200 μM dNTPs, 2.5 U Taq polymerase, 1.5 mM MgCl₂,15 pmole of each primer and 0.1 to 0.5 μg DNA. Nucleotide sequenceanalysis was performed by use of an automated DNA sequencer Model 373A(Applied Biosystems, Foster City, Calif., USA) withfluorescence-labelled dideoxy nucleotides (Prism™ Ready Reaction DyeTerminator Cycle Sequencing Kit; Applied Biosystems, Foster City,Calif., USA). The primers used for the sequencing reaction were the sameas for the amplification step. The following sequences, corresponding toexon 2 and exon 3 of the allele B*51new were obtained:GCTCCCACTCCATGAGGTATTTCTACACCGCCGTGTGCCGGCGGGGCCGCGGGGAGCCCCGC (SEQ IDNO 74) TTCATCTCAGTGGGCTACGTGGACGAGACGCAGTTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGAGAGGAGCCGCGGGGGCCGTGGATAGAGCAGGAGGGGCCGGAATATTGGGACCGGAACACACAGATCTGCAAGACCAACACACAGACTGACCGAGAGAGCCTGCGGAACCTGCGCGGCTACTACAACCAGAGCGAGGCCG        exon 2GGTCTCACACCCTCCAGAGGATGTACGGCTGCGACGTGGGGCCGGACGGGCGCCTCCTCCGC (SEQ IDNO 75) GGGTATAACCAGTTCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGAGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGAGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGACACCTGGAGAACGGGAAGGAGACGCTGCAGCGCGCGG       exon 3

Example 12 Typing of the Allele B*51new

[0189] The following method, that may be used to type the allele B*1406in a sample, is based on the LiPA technology (Stuyver et al, 1993).Nucleic acids are extracted from a blood sample with the QIAamp BloodKit, as indicated in example 11. For the amplification step the primerpair IBPIN1 (SEQ ID NO 26) and IBPIN3 (SEQ ID NO 27) is used. The PCRreaction is performed under the same conditions as in example 11. Theallele HLA-B*51new can subsequently be typed by a reverse hybridizationstep to a panel of oligonucleotide probes that are immobilized on anitro-cellulose strip. For instance, FIG. 6 shows the result of areverse hybidization assay according to the LiPA technique. After anamplification step as described above, the amplified nucleic acids werehybridized to a panel of 60 probes by use of the LiPA HLA-B kit(Innogenetics NV, Ghent, Belgium) according to the manufacturer'sprotocol. From the result shown in FIG. 6, it can be derived that thesample contained the allele HLA-B*51new, in combination with the knownallele HLA-B*1501. The numbers in FIG. 6 indicate probes that arepresent on the strip (note that these probes are not the same probes asthose in FIGS. 1-3). The amplified nucleic acid fragment of alleleHLA-B*51new hybridizes to the following probes (lines) on the strip: 3(SEQ ID NO 90), 6 (SEQ ID NO 91), 9 (SEQ ID NO 79), 14 (SEQ ID NO 92),18 (SEQ ID NO 93), 23 (SEQ ID NO 98), 31 (SEQ ID NO 87), 35 (SEQ ID NO99), 46 (SEQ ID NO 94), 49 (SEQ ID NO 95), 52 (SEQ ID NO 100), 55 (SEQID NO 89).

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1 107 1 269 DNA Homo sapiens 1 cacgtttctt ggagtactct acgtctgagtgtcatttctt caatgggacg gagcgggtgc 60 ggttcctgga cagatacttc tataaccaagaggagtacgt gcgcttcgac agcgacgtgg 120 gggagtaccg ggcggtgacg gagctggggcggcctgatgc cgagtactgg aacagccaga 180 aggacttcct ggaagacagg cgggccctggtggacaccta ctgcagacac aactacgggg 240 ttgtggagag cttcacagtg cagcggcga 2692 89 PRT Homo sapiens 2 Arg Phe Leu Glu Tyr Ser Thr Ser Glu Cys His PhePhe Asn Gly Thr 1 5 10 15 Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe TyrAsn Gln Glu Glu Tyr 20 25 30 Val Arg Phe Asp Ser Asp Val Gly Glu Tyr ArgAla Val Thr Glu Leu 35 40 45 Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser GlnLys Asp Phe Leu Glu 50 55 60 Asp Arg Arg Ala Leu Val Asp Thr Tyr Cys ArgHis Asn Tyr Gly Val 65 70 75 80 Val Glu Ser Phe Thr Val Gln Arg Arg 85 324 DNA Homo sapiens 3 gatccttcgt gtccccacag cacg 24 4 27 DNA Homosapiens 4 ccccacagca cgtttcttgg agtactc 27 5 24 DNA Homo sapiens 5tgtccccaca gcacgtttct tgtg 24 6 20 DNA Homo sapiens 6 tttcttggagcaggttaaac 20 7 25 DNA Homo sapiens 7 cacgtttctt gaagcaggat aagtt 25 820 DNA Homo sapiens 8 cacagcacgt ttcttggagg 20 9 18 DNA Homo sapiens 9ctgtggcagc ctaagagg 18 10 22 DNA Homo sapiens 10 tcgccgctgc actgtgaagctc 22 11 16 DNA Homo sapiens 11 attcccgcgc cgcgct 16 12 20 DNA Homosapiens 12 ctgcactgtg aagctctcca 20 13 18 DNA Homo sapiens 13 gtactctacgtctgagtg 18 14 19 DNA Homo sapiens 14 gcctgatgcc gagtactgg 19 15 19 DNAHomo sapiens 15 agaaggactt cctggaaga 19 16 21 DNA Homo sapiens 16agccaggcgg gccctggtgg a 21 17 15 DNA Homo sapiens 17 ggggttgtgg agagc 1518 18 DNA Homo sapiens 18 ttcttcaatg ggacggag 18 19 18 DNA Homo sapiens19 ttcctggaca gatacttc 18 20 18 DNA Homo sapiens 20 caagaggagt acgtgcgc18 21 18 DNA Homo sapiens 21 ggggagtacc gggcggtg 18 22 270 DNA Homosapiens 22 gctcccactc catgaggtat ttctacacct ccgtgtcccg gcccggccgcggggagcccc 60 gcttcatctc agtgggctac gtggacgaca cgcagttcgt gaggttcgacagcgacgccg 120 cgagtccgag agaggagccg cgggcgccgt ggatagagca ggaggggccggagtattggg 180 accgggagac acagatctcc aagaccaaca cacagactta ccgagagagcctgcggaacc 240 tgcgcggcta ctacaaccag agcgaggccg 270 23 276 DNA Homosapiens 23 ggtctcacac cctccagagg atgtacggct gcgacgtggg gccggacgggcgcctcctcc 60 gcgggcataa ccagttcgcc tacgacggca aggattacat cgccctgaacgaggacctga 120 gctcctggac cgcggcggac accgcggctc agatcaccca gcgcaagtgggaggcggccc 180 gtgtggcgga gcagctgaga acctacctgg agggcacgtg cgtggagtggctccgcagat 240 acctggagaa cgggaaggag acgctgcagc gcgcgg 276 24 90 PRTHomo sapiens 24 Gly Ser His Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser ArgPro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val AspAsp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg GluGlu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp AspArg Glu Thr 50 55 60 Gln Ile Ser Lys Thr Asn Thr Gln Thr Tyr Arg Glu SerLeu Arg Asn 65 70 75 80 Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala 85 90 2592 PRT Homo sapiens 25 Gly Ser His Thr Leu Gln Arg Met Tyr Gly Cys AspVal Gly Pro Asp 1 5 10 15 Gly Arg Leu Leu Arg Gly His Asn Gln Phe AlaTyr Asp Gly Lys Asp 20 25 30 Tyr Ile Ala Leu Asn Glu Asp Leu Ser Ser TrpThr Ala Ala Asp Thr 35 40 45 Ala Ala Gln Ile Thr Gln Arg Lys Trp Glu AlaAla Arg Val Ala Glu 50 55 60 Gln Leu Arg Thr Tyr Leu Glu Gly Thr Cys ValGlu Trp Leu Arg Arg 65 70 75 80 Tyr Leu Glu Asn Gly Lys Glu Thr Leu GlnArg Ala 85 90 26 22 DNA Homo sapiens 26 gggaggagcg aggggaccsc ac 22 2723 DNA Homo sapiens 27 ggaggccatc cccggcgacc tat 23 28 18 DNA Homosapiens 28 gggacacgga ggtgtaga 18 29 16 DNA Homo sapiens 29 ccggcccggccgcggg 16 30 18 DNA Homo sapiens 30 gcttcatctc agtgggct 18 31 17 DNAHomo sapiens 31 gttcgtgagg ttcgaca 17 32 19 DNA Homo sapiens 32gagtccgaga gaggagccg 19 33 17 DNA Homo sapiens 33 ggccggagta ttgggac 1734 18 DNA Homo sapiens 34 ggaccgggag acacagat 18 35 17 DNA Homo sapiens35 agatctccaa gaccaac 17 36 17 DNA Homo sapiens 36 cacagactta ccgagag 1737 17 DNA Homo sapiens 37 accgagagag cctgcgg 17 38 17 DNA Homo sapiens38 cggaacctgc gcggcta 17 39 17 DNA Homo sapiens 39 agaggatgta cggctgc 1740 16 DNA Homo sapiens 40 gacgtggggc cggacg 16 41 17 DNA Homo sapiens 41gacgggcgcc tcctccg 17 42 20 DNA Homo sapiens 42 tcctccgcgg gcataaccag 2043 19 DNA Homo sapiens 43 gggcataacc agttcgcct 19 44 18 DNA Homo sapiens44 gaggacctga gctcctgg 18 45 17 DNA Homo sapiens 45 cggcccgtgt ggcggag17 46 18 DNA Homo sapiens 46 gcagctgaga acctacct 18 47 17 DNA Homosapiens 47 tggagggcac gtgcgtg 17 48 16 DNA Homo sapiens 48 cgtggagtggctccgc 16 49 18 DNA Homo sapiens 49 tccgcagata cctggaga 18 50 243 DNAHomo sapiens 50 tttcttggag caggttaaac ctgagtgtca tttcttcaac gggacggagcgggtgcggtt 60 cctggacaga tacttctatc accaagagga gtacgtgcgc ttcgacagcgacgtggggga 120 gtaccgggcg gtgacggagc tggggcggcc tgatgccgag tactggaacagccagaagga 180 cctcctggag cagaagcggg ccgcggtgga cacctactgc agacacaactacggggttgg 240 tga 243 51 80 PRT Homo sapiens 51 Phe Leu Glu Gln Val LysPro Glu Cys His Phe Phe Asn Gly Thr Glu 1 5 10 15 Arg Val Arg Phe LeuAsp Arg Tyr Phe Tyr His Gln Glu Glu Tyr Val 20 25 30 Arg Phe Asp Ser AspVal Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly 35 40 45 Arg Pro Asp Ala GluTyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln 50 55 60 Lys Arg Ala Ala ValAsp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly 65 70 75 80 52 23 DNA Homosapiens 52 acgtttcttg gagcaggtta aac 23 53 24 DNA Homo sapiens 53accggatcct tcgtgtcccc acag 24 54 19 DNA Homo sapiens 54 taagtgtgagtgtcatttc 19 55 15 DNA Homo sapiens 55 cagaagcggg ccgcg 15 56 20 DNAHomo sapiens 56 atcaccaaga ggagtacgtg 20 57 20 DNA Homo sapiens 57cacaactacg gggttggtga 20 58 19 DNA Homo sapiens 58 gcctgatgcc gagtactgg19 59 15 DNA Homo sapiens 59 gccgcggtgg acacc 15 60 18 DNA Homo sapiens60 ccagaaggac ctcctgga 18 61 21 DNA Homo sapiens 61 cagatacttctatcaccaag a 21 62 15 DNA Homo sapiens 62 gggccgaggt ggaca 15 63 18 DNAHomo sapiens 63 ccagaaggac ctcctgga 18 64 15 DNA Homo sapiens 64ggggttgtgg agagc 15 65 16 DNA Homo sapiens 65 gaggagtacg cgcgct 16 66 16DNA Homo sapiens 66 gagcgagtgt ggaacc 16 67 270 DNA Homo sapiens 67cacgtttctt ggagcaggct aagtgtgagt gtcatttcct caatgggacg gagcgagtgt 60ggaacctgat cagatacatc tataaccaag aggagtacgc gcgctacaac agtgatctgg 120gggagtacca ggcggtgacg gagctggggc ggcctgacgc tgagtactgg aacagccaga 180aggacctcct ggagcggagg cgggccgagg tggacaccta ctgcagatac aactacgggg 240ttgtggagag cttcacagtg cagcggcgag 270 68 289 DNA Homo sapiens 68gatccttcgt gtccccacag cacgtttctt ggagcaggtt aaacctgagt gtcatttctt 60caacgggacg gagcgggtgc ggttcctgga cagatacttc tatcaccaag aggagtacgt 120gcgcttcgac agcgacgtgg gggagtaccg ggcggtgacg gagctggggc ggcctgatgc 180cgagtactgg aacagccaga aggacctcct ggagcagaag cgggccgcgg tggacaccta 240ctgcagacac aactacgggg ttggtgagag cttcacagtg cagcggcga 289 69 268 DNAHomo sapiens 69 acgtttcttg gagcaggtta aacctgagtg tcatttcttc aacgggacggagcgggtgcg 60 gttcctggac agatacttct atcaccaaga ggagtacgtg cgcttcgacagcgacgtggg 120 ggagtaccgg gcggtgacgg agctggggcg gcctgatgcc gagtactggaacagccagaa 180 ggacctcctg gagcagaagc gggccgcggt ggacacctac tgcagacacaactacggggt 240 tggtgagagc ttcacagtgc agcggcga 268 70 315 DNA Homosapiens 70 accggatcct tcgtgtcccc acagcacgtt tcttggagca ggctaagtgtgagtgtcatt 60 tcctcaatgg gacggagcga gtgtggaacc tgatcagata catctataaccaagaggagt 120 acgcgcgcta caacagtgat ctgggggagt accaggcggt gacggagctggggcggcctg 180 acgctgagta ctggaacagc cagaaggacc tcctggagcg gaggcgggccgaggtggaca 240 cctactgcag atacaactac ggggttgtgg agagcttcac agtgcagcggcgaggtgagc 300 atggtggagg gcggg 315 71 249 DNA Homo sapiens 71taagtgtgag tgtcatttcc tcaatgggac ggagcgagtg tggaacctga tcagatacat 60ctataaccaa gaggagtacg cgcgctacaa cagtgatctg ggggagtacc aggcggtgac 120ggagctgggg cggcctgacg ctgagtactg gaacagccag aaggacctcc tggagcggag 180gcgggccgag gtggacacct actgcagatc aactacgggg ttgtggagag cttcacagtg 240cagcggcga 249 72 270 DNA Homo sapiens 72 gctcccactc catgaggtatttctacaccg ccgtgtcccg gcccggccgc ggggagcccc 60 gcttcatctc agtgggctacgtggacgaca cgcagttcgt gaggttcgac agcgacgccg 120 cgagtccgag agaggagccgcgggcgccgt ggatagagca ggaggggccg gaatattggg 180 accggaacac acagatctgcaagaccaaca cacagactga ccgagagagc ctgcggaacc 240 tgcgcggcta ctacaaccagagcgaggccg 270 73 276 DNA Homo sapiens 73 ggtctcacac cctccagaggatgtacggct gcgacgtggg gccggacggg cgcctcctcc 60 gcgggtataa ccagttcgcctacgacggca aggattacat cgccctgaac gaggacctga 120 gctcctggac cgcggcggacaccgcggctc agatcaccca gcgcaagtgg gaggcggccc 180 gtgaggcgga gcagctgagagcctacctgg agggcacgtg cgtggagtgg ctccgcagac 240 acctggagaa cgggaaggagacgctgcagc gcgcgg 276 74 270 DNA Homo sapiens 74 gctcccactc catgaggtatttctacaccg ccatgtcccg gcccggccgc ggggagcccc 60 gcttcattgc agtgggctacgtggacgaca cccagttcgt gaggttcgac agcgacgccg 120 cgagtccgag gacggagccccgggcgccat ggatagagca ggaggggccg gagtattggg 180 accggaacac acagatcttcaagaccaaca cacagactta ccgagagaac ctgcggatcg 240 cgctccgcta ctacaaccagagcgaggccg 270 75 276 DNA Homo sapiens 75 ggtctcacac ttggcagacgatgtatggct gcgacgtggg gccggacggg cgcctcctcc 60 ccgggcataa ccagtacgcctacgacggca aagattacat cgccctgaac gaggacctga 120 gctcctggac cgcggcggacaccgcggctc agatcaccca gcgcaagtgg gaggcggccc 180 gtgaggcgga gcagctgagagcctacctgg agggcctgtg cgtggagtgg ctccgcagac 240 acctggagaa cgggaaggagtcgctgcagc gcgcgg 276 76 17 DNA Homo sapiens 76 tctacaccgc cgtgtcc 17 7718 DNA Homo sapiens 77 gcgacgccgc gagtccga 18 78 17 DNA Homo sapiens 78ggccggaata ttgggac 17 79 16 DNA Homo sapiens 79 ggaccggaac acacag 16 8017 DNA Homo sapiens 80 acagatctgc aagacca 17 81 16 DNA Homo sapiens 81acagactgac cgagag 16 82 19 DNA Homo sapiens 82 ggaacctgcg cggctacta 1983 16 DNA Homo sapiens 83 agaggatgta cggctg 16 84 17 DNA Homo sapiens 84cgacgtgggg ccggacg 17 85 19 DNA Homo sapiens 85 gggtataacc agttcgcct 1986 17 DNA Homo sapiens 86 aggacctgag ctcctgg 17 87 16 DNA Homo sapiens87 gcccgtgagg cggagc 16 88 18 DNA Homo sapiens 88 gcagctgaga gcctacct 1889 17 DNA Homo sapiens 89 ccgcagacac ctggaga 17 90 18 DNA Homo sapiens90 gcttcattgc agtgggct 18 91 23 DNA Homo sapiens 91 cgagtccgaggacggagccc cgg 23 92 18 DNA Homo sapiens 92 cagatcttca agaccaac 18 93 19DNA Homo sapiens 93 cacacagact taccgagag 19 94 18 DNA Homo sapiens 94cgagagaacc tgcggatc 18 95 17 DNA Homo sapiens 95 cggatcgcgc tccgcta 1796 17 DNA Homo sapiens 96 tctacaccgc catgtcc 17 97 17 DNA Homo sapiens97 gcgacgccgc gagtccg 17 98 17 DNA Homo sapiens 98 acacttggca gacgatg 1799 16 DNA Homo sapiens 99 ggagggcctg tgcgtg 16 100 19 DNA Homo sapiens100 cataaccagt acgcctacg 19 101 15 DNA Homo sapiens 101 gtggagtggc tccgc15 102 16 DNA Homo sapiens 102 gacgggcgcc tcctcc 16 103 90 PRT Homosapiens 103 Gly Ser His Ser Met Arg Tyr Ser Tyr Thr Ala Val Ser Arg ProGly 1 5 10 15 Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val Asp AspThr Gln 20 25 30 Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Glu GluPro Arg 35 40 45 Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp ArgAsn Thr 50 55 60 Gln Ile Cys Lys Thr Asn Thr Gln Thr Asp Arg Glu Ser LeuArg Asn 65 70 75 80 Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala 85 90 104 92PRT Homo sapiens 104 Gly Ser His Thr Leu Gln Arg Met Tyr Gly Cys Asp ValGly Pro Asp 1 5 10 15 Gly Arg Leu Leu Arg Gly Tyr Asn Gln Phe Ala TyrAsp Gly Lys Asp 20 25 30 Tyr Ile Ala Leu Asn Glu Asp Leu Ser Ser Trp ThrAla Ala Asp Thr 35 40 45 Ala Ala Gln Ile Thr Gln Arg Lys Trp Glu Ala AlaArg Glu Ala Glu 50 55 60 Gln Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val GluTrp Leu Arg Arg 65 70 75 80 His Leu Glu Asn Gly Lys Glu Thr Leu Gln ArgAla 85 90 105 90 PRT Homo sapiens 105 Gly Ser His Ser Met Arg Tyr PheTyr Thr Ala Met Ser Arg Pro Gly 1 5 10 15 Arg Gly Glu Pro Arg Phe IleAla Val Gly Tyr Val Asp Asp Thr Gln 20 25 30 Phe Val Arg Phe Asp Ser AspAla Ala Ser Pro Arg Thr Glu Pro Arg 35 40 45 Ala Pro Trp Ile Glu Gln GluGly Pro Glu Tyr Trp Asp Arg Asn Thr 50 55 60 Gln Ile Phe Lys Thr Asn ThrGln Thr Tyr Arg Glu Asn Leu Arg Ile 65 70 75 80 Ala Leu Arg Tyr Tyr AsnGln Ser Glu Ala 85 90 106 92 PRT Homo sapiens 106 Gly Ser His Thr TrpGln Thr Met Tyr Gly Cys Asp Val Gly Pro Asp 1 5 10 15 Gly Arg Leu LeuPro Gly His Asn Gln Tyr Ala Tyr Asp Gly Lys Asp 20 25 30 Tyr Ile Ala LeuAsn Glu Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr 35 40 45 Ala Ala Gln IleThr Gln Arg Lys Trp Glu Ala Ala Arg Glu Ala Glu 50 55 60 Gln Leu Arg AlaTyr Leu Glu Gly Leu Cys Val Glu Trp Leu Arg Arg 65 70 75 80 His Leu GluAsn Gly Lys Glu Ser Leu Gln Arg Ala 85 90 107 21 DNA Homo sapiens 107cccgccctcc accatgctca c 21

1. A method for determining the presence or absence of an alleleselected from the group consisting of HLA-B*3913, HLA-B*1406 andHLA-B*51new in a sample, wherein exon 2 of said alleles is defined bySEQ ID NO: 22, 72 and 74 respectively, and exon 3 of said allclos isdefined by SEQ ID NO: 23, 73 and 75 respectively, wherein said methodcomprises: a) obtaining nucleic acids from the sample, b) amplifyingnucleic acids comprising part or all of exon 2 and/or part or all ofexon 3 of said allele obtained from the sample using at least onesuitable pair of primers, and c) analyzing me amplified nucleic acidsfor the presence or absence of HLA-B*3913, HLA B*1406 find/or HLA-B*51₅respectively.
 2. The method according to claim 1, wherein the analysisof the amplified nucleic acids comprises: a) hybridizing the amplifiednucleic acids to a set of probes which specifically hybridize to targetregions within the nucleic adds, which regions comprise one or morepolymorphic nucleotides in exon 2 and/or exon 3 of said allele; and b)determining the absence or presence of the allele HLA-B*3913, HLA-B*1406and/or HLA-B*51new.
 3. The method according to claim 2, wherein said oneor more polymorphic nucleotides has a position within exon 2 selectedfrom the group consisting of nucleoude positions: 11, 24, 30, 33, 44,46, 68, 69, 71, 88, 92, 94, 102, 120, 131, 132, 133, 136, 140, 149, 153,155, 161, 173, 174, 183, 186, 188, 190, 193, 196, 197, 198, 199, 200,204, 205, 207, 208, 209, 210, 212, 219, 226, 228, 229, 236, 238, 240,241, 244, 246 and 268, and/or a position within exon 3 selected from thegroup consisting of nucleotide positions: 2, 10, 11, 12, 13, 14, 18, 19,20, 26, 36, 44, 54, 66, 68, 69, 75, 76, 77, 92, 120, 134, 141, 142, 145,156, 159, 163, 169, 184, 195, 196, 197, 201, 214, 216, 217, 227, 22S,229 and
 240. 4. The method according to claim 1, wherein said primerscomprise a nucleotide sequence selected from the group consisting of SEQID NO: 26 and
 27. 5. The method according to claim 2, wherein saidprobes comprise a nucleotide sequence selected from the group consistingof: SEQ ID NO: 28-49 for the typing of HLA-B*3913: SEQ ID NO 29, 30, 32,37, 41, 47, 48 and 16-89 for the typing of HLA-B*1406; and SEQ ID NO 33,44, 79, 84, 87 and 89-102 for the typing of HLA-B*51new.
 6. An isolatednucleic acid comprising exon 2 and/or exon 3 of the allele HLA-B*3913,HLA-B*1406 or HLA-B*51new.
 7. An isolated nucleic acid comprising asequence selected from the group consisting of: SEQ ID NO: 22, SEQ IDNO: 72, SEQ ID NO: 74, SEQ ID NO: 23, SEQ ID NO: 73 and SEQ ID NO: 75.8. A diagnostic kit for the typing of the alleles HLA-B-i-SQIS,HLA.-B+1406 and/or BLA-B*51 comprising the nucleic acid of claim 7.